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Abstract:

The present invention provides improved methods for the manufacturing of
IVIG products. These methods offer various advantages such as reduced
loss of IgG during purification and improved quality of final products.
In other aspects, the present invention provides aqueous and
pharmaceutical compositions suitable for intravenous, subcutaneous,
and/or intramuscular administration. In yet other embodiments, the
present invention provides methods of treating a disease or condition
comprising administration of an IgG composition provided herein.

Claims:

1-58. (canceled)

59. A method for preparing an enriched IgG composition from plasma, the
method comprising the steps of: (a) precipitating a cryo-poor plasmid
fraction with between about 6% and about 10% alcohol at a pH of between
about 6.7 and about 7.3 to obtain a first precipitate and a first
supernatant enriched in IgG; (b) precipitating IgG from the first
supernatant with between about 20% and about 30% alcohol at a temperature
between about -5.degree. C. and about -9.degree. C. and at a pH of
between about 6.7 and about 7.1 to form a second precipitate; (c)
re-suspending the second precipitate to form a suspension; (d) treating
the suspension with finely divided silicon dioxide (SiO2); and (e)
separating the soluble fraction from the suspension treated with finely
divided silicon dioxide, thereby forming an enriched IgG composition.

60. The method of claim 59, wherein IgG is precipitated from the first
supernatant in step (b) with between about 23% and about 27% alcohol.

61. The method of claim 59, wherein IgG is precipitated from the first
supernatant in step (b) with between about 24% and about 26% alcohol.

62. The method of claim 59, wherein IgG is precipitated from the first
supernatant in step (b) with about 25% alcohol.

63. The method of claim 59, wherein IgG is precipitated from the first
supernatant in step (b) at a temperature between about -7.degree. C. and
about -9.degree. C.

64. The method of claim 59, wherein IgG is precipitated from the first
supernatant in step (b) at a temperature of about -7.degree. C.

65. The method of claim 59, wherein IgG is precipitated from the first
supernatant in step (b) at a pH of about 6.9.

66. The method of claim 59, wherein IgG is precipitated from the first
supernatant in step (b) with between about 23% and about 27% alcohol at a
temperature between about -7.degree. C. and about -9.degree. C.

67. The method of claim 66, wherein IgG is precipitated from the first
supernatant in step (b) with about 25% alcohol at a temperature of about
-7.degree. C.

68. The method of claim 66, wherein IgG is precipitated from the first
supernatant in step (b) at a pH of about 6.9.

69. The method of claim 67, wherein IgG is precipitated from the first
supernatant in step (b) at a pH of about 6.9.

70. The method of claim 59, wherein the second precipitate is
re-suspended with an extraction buffer at a ratio of 1 part precipitate
to between about 8 and about 30 parts extraction buffer.

71. The method of claim 59, wherein the second precipitate is
re-suspended in step (c) with an extraction buffer at a ratio of 1 part
precipitate to about 15 parts extraction buffer.

72. The method of claim 59, wherein the second precipitate is
re-suspended in step (c) with an extraction buffer comprising monobasic
sodium phosphate and acetate.

73. The method of claim 72, wherein the extraction buffer has a pH of
about 4.5.+-.0.2.

75. The method of claim 74, wherein the pH of the extraction buffer is
adjusted with between 400 mL and 700 mL of glacial acetic acid per 1000 L
of buffer.

76. The method of claim 75, wherein the pH of the extraction buffer is
adjusted with between 510 mL and 600 mL of glacial acetic acid per 1000 L
of buffer.

77. The method of claim 66, wherein the extraction buffer comprises 5 mM
monobasic sodium phosphate and 5 mM acetate and wherein the pH of the
extraction buffer is adjusted with between 510 mL and 600 mL of glacial
acetic acid per 1000 L of buffer.

78. The method of claim 67, wherein the extraction buffer comprises 5 mM
monobasic sodium phosphate and 5 mM acetate and wherein the pH of the
extraction buffer is adjusted with between 510 mL and 600 mL of glacial
acetic acid per 1000 L of buffer.

79. The method of claim 68, wherein the extraction buffer comprises 5 mM
monobasic sodium phosphate and 5 mM acetate and wherein the pH of the
extraction buffer is adjusted with between 510 mL and 600 mL of glacial
acetic acid per 1000 L of buffer.

80. The method of claim 69, wherein the extraction buffer comprises 5 mM
monobasic sodium phosphate and 5 mM acetate and wherein the pH of the
extraction buffer is adjusted with between 510 mL and 600 mL of glacial
acetic acid per 1000 L of buffer.

81. The method of claim 59, wherein treating the suspension with finely
divided silicon dioxide (SiO2) in step (d) comprises the addition of
between about 0.01 g and about 0.07 g fumed silicon per g of the
precipitate formed in step (b).

82. The method of claim 59, wherein treating the suspension with finely
divided silicon dioxide (SiO2) in step (d) comprises the addition of
between about 0.02 g and about 0.06 g fumed silicon per g of the
precipitate formed in step (b).

83. The method of claim 59, wherein treating the suspension with finely
divided silicon dioxide (SiO2) in step (d) comprises the addition of
between about 0.03 g and about 0.05 g fumed silicon per g of the
precipitate formed in step (b).

84. The method of claim 66, wherein treating the suspension with finely
divided silicon dioxide (SiO2) in step (d) comprises the addition of
between about 0.03 g and about 0.05 g fumed silicon per g of the
precipitate formed in step (b).

85. The method of claim 67, wherein treating the suspension with finely
divided silicon dioxide (SiO2) in step (d) comprises the addition of
between about 0.03 g and about 0.05 g fumed silicon per g of the
precipitate formed in step (b).

86. The method of claim 68, wherein treating the suspension with finely
divided silicon dioxide (SiO2) in step (d) comprises the addition of
between about 0.03 g and about 0.05 g fumed silicon per g of the
precipitate formed in step (b).

87. The method of claim 69, wherein treating the suspension with finely
divided silicon dioxide (SiO2) in step (d) comprises the addition of
between about 0.03 g and about 0.05 g fumed silicon per g of the
precipitate formed in step (b).

88. The method of claim 59, wherein treating the suspension with finely
divided silicon dioxide (SiO2) in step (d) comprises the addition of
about 0.04 g fumed silicon per g of the precipitate formed in step (b).

89. The method of claim 66, wherein treating the suspension with finely
divided silicon dioxide (SiO2) in step (d) comprises the addition of
about 0.04 g fumed silicon per g of the precipitate formed in step (b).

90. The method of claim 67, wherein treating the suspension with finely
divided silicon dioxide (SiO2) in step (d) comprises the addition of
about 0.04 g fumed silicon per g of the precipitate formed in step (b).

91. The method of claim 68, wherein treating the suspension with finely
divided silicon dioxide (SiO2) in step (d) comprises the addition of
about 0.04 g fumed silicon per g of the precipitate formed in step (b).

92. The method of claim 69, wherein treating the suspension with finely
divided silicon dioxide (SiO2) in step (d) comprises the addition of
about 0.04 g fumed silicon per g of the precipitate formed in step (b).

93. The method of claim 59, wherein treating the suspension with finely
divided silicon dioxide (SiO2) in step (d) comprises incubating the
suspension for at least 30 minutes in the presence of the finely divided
silicon dioxide particles.

94. The method of claim 59, wherein the treatment with finely divided
silicon dioxide (SiO2) in step (d) is performed at a temperature
between about 2.degree. C. and about 10.degree. C.

96. The method of claim 95, wherein the depth filtration further
comprises purging the filter with at least 3 dead volumes of buffer.

97. The method of claim 96, wherein the depth filtration further
comprises purging the filter with at least 3.6 dead volumes of buffer.

98. The method of claim 59, wherein the method further comprises a step
of precipitating IgG in a third precipitation step with between about 22%
and about 28% alcohol at a pH of between about 6.7 and about 7.3 to form
a third precipitate.

99. The method of claim 59, wherein the method further comprises a
solvent detergent (S/D) treatment.

101. The method of claim 100, wherein the method comprises both an anion
exchange chromatography purification step and a cation exchange
chromatography step.

102. The method of claim 59, wherein the method further comprises a
nanofiltration step and/or an ultrafiltration/diafiltration step.

103. The method of claim 59, wherein the enriched IgG composition
obtained in step (e) contains at least 85% of the IgG content found in
the cryo-poor plasma fraction used in step (a).

104. The method of claim 59, wherein the enriched IgG composition
obtained in step (e) contains at least 90% of the IgG content found in
the cryo-poor plasma fraction used in step (a).

105. An aqueous IgG composition prepared according to the method of claim
59.

106. The aqueous IgG composition of claim 105, wherein the composition
comprises at least about 80 grams of protein per liter of the
composition.

107. The aqueous IgG composition of claim 105, wherein at least 95% of
the protein is IgG.

108. The aqueous IgG composition of claim 105, wherein at least 98% of
the protein is IgG.

109. The aqueous IgG composition of claim 105, wherein the composition
contains less than about 35 μg/mL IgA.

110. A pharmaceutical composition comprising an aqueous IgG composition
prepared according to the method of claim 59.

111. The pharmaceutical composition of claim 110, wherein the composition
comprises about 100 grams of protein per liter of the composition.

112. The pharmaceutical composition of claim 110, wherein the composition
further comprises glycine at a concentration from between about 200 mM to
about 300 mM.

113. The pharmaceutical composition of claim 110, wherein the pH of the
composition is between about 4.6 and about 5.1.

114. The pharmaceutical composition of claim 110, wherein the osmolarity
of the composition is between about 240 mOsmol/kg and about 300
mOsmol/kg.

115. The pharmaceutical composition of claim 110, wherein the composition
is stable at room temperature for at least about 9 months.

116. The pharmaceutical composition of claim 110, wherein the composition
is stable at a temperature between about 2.degree. C. and about 8.degree.
C. for at least about 36 months.

117. The pharmaceutical composition of claim 110, wherein the composition
is formulated for intravenous administration.

118. A method of treating an immunodeficiency, autoimmune disease, or
acute infection in a human in need thereof, the method comprising
administering a pharmaceutical composition of claim 110.

Description:

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to an Australian application,
entitled "A Method to Produce an Immunoglobulin Preparation with Improved
Yield," filed May 26, 2010 (Attorney Docket No. 20695C-021300AU), which
is hereby incorporated herein by reference in its entirety for all
purposes.

BACKGROUND OF THE INVENTION

[0002] Immune globulin products from human plasma were first used in 1952
to treat immune deficiency. Initially, intramuscular or subcutaneous
administration of Immunoglobulin isotype G (IgG) were the methods of
choice. For injecting larger amounts of IgG necessary for effective
treatment of various diseases, however, the intravenous administrable
products with lower concentrated IgG (50 mg/mL) were developed. Usually
intravenous immunoglobulin (IVIG), contains the pooled immunoglobulin G
(IgG) immunoglobulins from the plasma of more than a thousand blood
donors. Typically containing more than 95% unmodified IgG, which has
intact Fc-dependent effector functions, and only trace amounts of
immunoglobulin A (IgA) or immunoglobulin M (IgM), IVIGs are sterile,
purified IgG products primarily used in treating three main categories of
medical conditions: (1) immune deficiencies such as X-linked
agammaglobulinemia, hypogammaglobulinemia (primary immune deficiencies),
and acquired compromised immunity conditions (secondary immune
deficiencies), featuring low antibody levels; (2) inflammatory and
autoimmune diseases; and (3) acute infections.

[0004] While IVIG treatment can be very effective for managing primary
immunodeficiency disorders, this therapy is only a temporary replacement
for antibodies that are not being produced in the body, rather than a
cure for the disease. Accordingly, patients dependent upon IVIG therapy
require repeated doses, typically about once a month for life. This need
places a great demand on the continued production of IVIG compositions.
However, unlike other biologics that are produced via in vitro expression
of recombinant DNA vectors, IVIG is fractionated from human blood and
plasma donations. Thus, IVIG products cannot be increased by simply
increasing the volume of production. Rather the level of commercially
available IVIG is limited by the available supply of blood and plasma
donations.

[0005] Several factors drive the demand for IVIG, including the acceptance
of IVIG treatments, the identification of additional indications for
which IVIG therapy is effective, and increasing patient diagnosis and
IVIG prescription. Notably, the global demand for IVIG has more than
quadrupled since 1990 and continues to increase today at an annual rate
between about 7% and 10% (Robert P., Pharmaceutical Policy and Law, 11
(2009) 359-367). For example, the Australian National Blood Authority
reported that the demand for IVIG in Australia grew by 10.6% for the
2008-2009 fiscal year (National Blood Authority Australia Annual Report
2008-2009).

[0006] Due in part to the increasing global demand and fluctuations in the
available supply of immunoglobulin products, several countries, including
Australia and England, have implemented demand management programs to
protect supplies of these products for the highest demand patients during
times of product shortages.

[0007] It has been reported that in 2007, 26.5 million liters of plasma
were fractionated, generating 75.2 metric tons of IVIG, with an average
production yield of 2.8 grams per liter (Robert P., supra). This same
report estimated that global IVIG yields are expected to increase to
about 3.43 grams per liter by 2012. However, due to the continued growth
in global demand for IVIG, projected at between about 7% and 13% annually
between now and 2015, further improvement of the overall IVIG yield will
be needed to meet global demand.

[0008] A number of IVIG preparation methods are used by commercial
suppliers of IVIG products. One common problem with the current IVIG
production methods is the substantial loss of IgG during the purification
process, estimated to be at least 30% to 35% of the total IgG content of
the starting material. One challenge is to maintain the quality of viral
inactivation and lack of impurities which can cause adverse reactions,
while bolstering the yield of IgG. At the current production levels of
IVIG, what may be considered small increases in the yield are in fact
highly significant. For example at 2007 production levels, a 2% increase
in efficiency, equal to an additional 56 milligrams per liter, would
generate 1.5 additional metric tons of IVIG.

[0009] In the fourth installment of a series of seminal papers published
on the preparation and properties of serum and plasma proteins, Cohn et
al. (J. Am. Chem. Soc., 1946, 68(3): 459-475) first described a methods
for the alcohol fractionation of plasma proteins (method 6), which allows
for the isolation of a fraction enriched in IgG from human plasma.
Several years later, Oncley et al. (J. Am. Chem. Soc., 1949, 71(2):
541-550) expanded upon the Cohn methods by publishing a method (method 9)
that resulted in the isolation of a purer IgG preparation.

[0010] These methods, while laying the foundation for an entire industry
of plasma derived blood factors, were unable to provide IgG preparations
having sufficiently high concentrations for the treatment of several
immune-related diseases, including Kawasaki syndrome, immune
thrombocytopenic purpura, and primary immune deficiencies. As such,
additional methodologies employing various techniques, such as ion
exchange chromatography, were developed to provide higher purity and
higher concentration IgG formulations. Hoppe et al. (Munch Med Wochenschr
1967 (34): 1749-1752) and Falksveden (Swedish Patent No. 348942) and
Falksveden and Lundblad (Methods of Plasma Protein Fractionation 1980)
were among the first to employ ion exchange chromatography for this
purpose.

[0011] Various modern methods employ a precipitation step, such as
caprylate precipitation (Lebing et al., Vox Sang 2003 (84):193-201) and
Cohn Fraction (I+)II+III ethanol precipitation (Tanaka et al., Braz J Med
Biol Res 2000 (33)37-30) coupled to column chromatography. Most recently,
Teschner et al. (Vox Sang, 2007 (92):42-55) have described a method for
production of a 10% IVIG product in which cryo-precipitate is first
removed from pooled plasma and then a modified Cohn-Oncley cold ethanol
fractionation is performed, followed by S/D treatment of the
intermediate, ion exchange chromatography, nanofiltration, and optionally
ultrafiltration/diafiltration.

[0012] However, despite the improved purity, safety, and yield afforded by
these IgG manufacturing methods, a significant amount of IgG is still
lost during the purification process. For example, Teschner et al. report
that their method results in an increased IgG yield of 65% (Teschner et
al., supra). As reported during various plasma product meetings, the
average yields for large-scale preparation of IgG, such as from Baxter,
CSL Behring, Upfront Technology, Cangene, Prometric BioTherapeutics, and
the Finnish Red Cross, range from about 61% to about 65% in the final
container. This represents a loss of at least about a third of the IgG
present in the pooled plasma fraction during the manufacturing process.

[0013] As such, a need exists for improved and more efficient methods for
manufacturing IVIG products. The present invention satisfies these and
other needs by providing IVIG manufacturing methods that produce yields
that are at least 6 to 10% higher than currently achievable, as well as
IVIG compositions provided there from.

BRIEF SUMMARY OF THE INVENTION

[0014] In one aspect, the present invention provides methods for preparing
an enriched IgG compositions (e.g., IVIG compositions) from plasma.
Advantageously, the methods provided herein provide significant
improvements over current state of the art manufacturing methods for
preparing IVIG compositions. For example, the methods provided herein
allow for increased yields of IgG in the final bulk composition without
losing the purity required for intravenous administration.

[0015] In one aspect, a method is provided for preparing an enriched IgG
composition from plasma comprising the steps of (a) precipitating a
cryo-poor plasma fraction, in a first precipitation step, with between
about 6% and about 10% alcohol at a pH of between about 7.0 and about 7.5
to obtain a first precipitate and a first supernatant, (b) precipitating
IgG from the first supernatant, in a second precipitation step, with
between about 20% and about 25% alcohol at a pH of between about 6.7 and
about 7.3 to form a second precipitate, (c) re-suspending the second
precipitate to form a suspension, (d) precipitating IgG from the
suspension formed in step (c), in a third precipitation step, with
between about 22% and about 28% alcohol at a pH of between about 6.7 and
about 7.3 to form a third precipitate, (e) re-suspending the third
precipitate to form a suspension, and (f) separating the soluble fraction
from the suspension formed in step (e), thereby forming an enriched IgG
composition, wherein at least one of the first precipitation step, second
precipitation step, or third precipitation step comprises spray addition
of the alcohol. In one embodiment, alcohol is added in the first
precipitation step by spraying. In another embodiment, alcohol is added
in the second precipitation step by spraying. In yet another embodiment,
alcohol is added in the third precipitation step by spraying.

[0016] In certain embodiments, the pH of one or more solution may be
adjusted by the addition of a pH modifying agent by spraying. In related
embodiments, the pH of at least one of the first precipitation step,
second precipitation step, or third precipitation step is achieved by
addition of a pH modifying solution after addition of the alcohol, or
before and after the addition of alcohol, during and after the addition
of alcohol, or before, during, and after the addition of alcohol. In yet
another related embodiment, the pH of a precipitation step may be
maintained for the entirety of the precipitation reaction by continuously
adjusting the pH.

[0017] In one specific embodiment, the pH of the first precipitation step
is adjusted after the addition of alcohol by spray addition of a pH
modifying agent. In another embodiment, the pH of the second
precipitation step is adjusted after the addition of alcohol by spray
addition of a pH modifying agent. In yet another embodiment, the pH of
the third precipitation step is adjusted after the addition of alcohol by
spray addition of a pH modifying agent.

[0018] Additionally, the preparatory methods provided herein may further
comprise an ion exchange chromatography step (i.e., anion exchange and/or
cation exchange chromatography), a nanofiltration step, an
ultrafiltration/diafiltration step, or any other suitable purification
technique to further enhance the purity or quality of the IVIG
preparations.

[0019] In another aspect, a method is provided for preparing an enriched
IgG composition from plasma comprising the steps of adjusting the pH of a
cryo-poor plasma fraction to at or about 7.0, (b) adjusting the ethanol
concentration of the cryo-poor plasma fraction of step (a) to at or about
25% (v/v) at a temperature between at or about -7° C. and at or
about -9° C., thereby forming a mixture, (c) separating liquid and
precipitate from the mixture of step (b), (d) re-suspending the
precipitate of step (c) with a buffer containing phosphate and acetate,
wherein the pH of the buffer is adjusted with at or about 600 ml of
glacial acetic acid per 1000 L of buffer, thereby forming a suspension,
(e) mixing finely divided silicon dioxide (SiO2) with the suspension
from step (d) for at least about 30 minutes, (f) filtering the suspension
with a filter press, thereby forming a filtrate, (g) washing the filter
press with at least 3 filter press dead volumes of a buffer containing
phosphate and acetate, wherein the pH of the buffer is adjusted with at
or about 150 ml of glacial acetic acid per 1000 L of buffer, thereby
forming a wash solution, (h) combining the filtrate of step (f) with the
wash solution of step (g), thereby forming a solution, and treating the
solution with a detergent, (i) adjusting the pH of the solution of step
(h) to at or about 7.0 and adding ethanol to a final concentration of at
or about 25%, thereby forming a precipitate, (j) separating liquid and
precipitate from the mixture of step (i), (k) dissolving the precipitate
in an aqueous solution comprising a solvent or detergent and maintaining
the solution for at least 60 minutes, (l) passing the solution after step
(k) through a cation exchange chromatography column and eluting proteins
absorbed on the column in an eluate, (m) passing the eluate from step (l)
through an anion exchange chromatography column to generate an effluent,
(n) passing the effluent from step (m) through a nanofilter to generate a
nanofiltrate, (o) passing the nanofiltrate from step (n) through an
ultrafiltration membrane to generate an ultrafiltrate; and (p)
diafiltrating the ultrafiltrate from step (o) against a diafiltration
buffer to generate a diafiltrate having a protein concentration between
about 8% (w/v) and about 12% (w/v), thereby obtaining a composition of
concentrated IgG.

[0020] In another aspect, the present invention provides aqueous IgG
compositions prepared by the methods described herein. Generally, the IgG
compositions have high purity (e.g., at least 95%, 98%, 99%, or higher
IgG contents), contain protein concentrations between about 20 g/L and
about 200 g/L, and contain extremely low levels of common IVIG
contaminants, such as IgG, IgM, Fibrinogen, Transferrin, ACA, amidolytic
activity, PKA, and the like.

[0021] In yet another aspect, pharmaceutical IgG compositions and
formulations suitable for use in IVIG therapies are provided. The
pharmaceutical formulations have high purity (e.g., at least 98%, 99%, or
higher IgG contents), contain protein concentrations between about 20 g/L
and about 200 g/L, and contain extremely low levels of common IVIG
contaminants, such as IgG, IgM, Fibrinogen, Transferrin, ACA, amidolytic
activity, PKA, and the like. Generally, the pharmaceutical compositions
are appropriately formulated for intravenous administration (i.e., for
IVIG therapy), subcutaneous administration, or intramuscular
administration.

[0023] FIG. 1: IgG concentration as determined by ELISA (.tangle-solidup.)
and nephelometric ( ) methods and total protein concentration
(.box-solid.) present in the Fraction II+III filtrate wash as a function
of the number of dead volumes of buffer used to wash the filtration
device post-filtration.

[0024] FIG. 2: Average PKA activity in Precipitate G dissolved fractions
after extraction and clarification at pH 3.8 to 5.0 by addition of acetic
acid in the presence and absence of Aerosil (silicon dioxide) treatment.

[0025]FIG. 3: Average fibrinogen content in Precipitate G dissolved
fractions after extraction and clarification at pH 3.8 to 5.0 by addition
of acetic acid in the presence and absence of Aerosil (silicon dioxide)
treatment.

[0026] FIG. 4: Average amidolytic activity in Precipitate G dissolved
fractions after extraction and clarification at pH 3.8 to 5.0 by addition
of acetic acid in the presence and absence of Aerosil (silicon dioxide)
treatment.

[0027] FIG. 5: Amidolytic activity in Precipitate G dissolved fractions
extracted and clarified at pH 3.8 to 7.8 after incubation for two weeks
at 4° C. (.diamond-solid.) or for an additional week at room
temperature (.box-solid.).

[0033] As used herein, an "antibody" refers to a polypeptide substantially
encoded by an immunoglobulin gene or immunoglobulin genes, or fragments
thereof, which specifically bind and recognize an analyte (antigen). The
recognized immunoglobulin genes include the kappa, lambda, alpha, gamma,
delta, epsilon and mu constant region genes, as well as the myriad
immunoglobulin variable region genes. Light chains are classified as
either kappa or lambda. Heavy chains are classified as gamma, mu, alpha,
delta, or epsilon, which in turn define the immunoglobulin classes, IgG,
IgM, IgA, IgD, and IgE, respectively.

[0034] An exemplary immunoglobulin (antibody) structural unit is composed
of two pairs of polypeptide chains, each pair having one "light" (about
25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino acids
primarily responsible for antigen recognition. The terms variable light
chain (VL) and variable heavy chain (VH) refer to these light
and heavy chains respectively.

[0035] As used herein, the term "ultrafiltration (UF)" encompasses a
variety of membrane filtration methods in which hydrostatic pressure
forces a liquid against a semi-permeable membrane. Suspended solids and
solutes of high molecular weight are retained, while water and low
molecular weight solutes pass through the membrane. This separation
process is often used for purifying and concentrating macromolecular
(103-106 Da) solutions, especially protein solutions. A number
of ultrafiltration membranes are available depending on the size of the
molecules they retain. Ultrafiltration is typically characterized by a
membrane pore size between 1 and 1000 kDa and operating pressures between
0.01 and 10 bar, and is particularly useful for separating colloids like
proteins from small molecules like sugars and salts.

[0036] As used herein, the term "diafiltration" is performed with the same
membranes as ultrafiltration and is a tangential flow filtration. During
diafiltration, buffer is introduced into the recycle tank while filtrate
is removed from the unit operation. In processes where the product is in
the retentate (for example IgG), diafiltration washes components out of
the product pool into the filtrate, thereby exchanging buffers and
reducing the concentration of undesirable species.

[0037] As used herein, the term "about" denotes an approximate range of
plus or minus 10% from a specified value. For instance, the language
"about 20%" encompasses a range of 18-22%.

[0038] As used herein, the term "mixing" describes an act of causing equal
distribution of two or more distinct compounds or substances in a
solution or suspension by any form of agitation. Complete equal
distribution of all ingredients in a solution or suspension is not
required as a result of "mixing" as the term is used in this application.

[0039] As used herein, the term "solvent" encompasses any liquid substance
capable of dissolving or dispersing one or more other substances. A
solvent may be inorganic in nature, such as water, or it may be an
organic liquid, such as ethanol, acetone, methyl acetate, ethyl acetate,
hexane, petrol ether, etc. As used in the term "solvent detergent
treatment," solvent denotes an organic solvent (e.g., tri-N-butyl
phosphate), which is part of the solvent detergent mixture used to
inactivate lipid-enveloped viruses in solution.

[0041] As used herein, the term "Intravenous IgG" or "IVIG" treatment
refers generally to a therapeutic method of intravenously,
subcutaneously, or intramuscularly administering a composition of IgG
immunoglobulins to a patient for treating a number of conditions such as
immune deficiencies, inflammatory diseases, and autoimmune diseases. The
IgG immunoglobulins are typically pooled and prepared from plasma. Whole
antibodies or fragments can be used. IgG immunoglobulins can be
formulated in higher concentrations (e.g., greater than 10%) for
subcutaneous administration, or formulated for intramuscular
administration. This is particularly common for specialty IgG
preparations which are prepared with higher than average titres for
specific antigens (e.g., Rho D factor, pertussis toxin, tetanus toxin,
botulism toxin, rabies, etc.). For ease of discussion, such
subcutaneously or intramuscularly formulated IgG compositions are also
included in the term "IVIG" in this application.

[0042] By "therapeutically effective amount or dose" or
"sufficient/effective amount or dose," it is meant a dose that produces
effects for which it is administered. The exact dose will depend on the
purpose of the treatment, and will be ascertainable by one skilled in the
art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage
Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of
Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);
and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,
Gennaro, Ed., Lippincott, Williams & Wilkins).

[0043] As used in this application, the term "spraying" refers to a means
of delivering a liquid substance into a system, e.g., during an alcohol
precipitation step, such as a modified Cohn fractionation I or II+III
precipitation step, in the form of fine droplets or mist of the liquid
substance. Spraying may be achieved by any pressurized device, such as a
container (e.g., a spray bottle), that has a spray head or a nozzle and
is operated manually or automatically to generate a fine mist from a
liquid. Typically, spraying is performed while the system receiving the
liquid substance is continuously stirred or otherwise mixed to ensure
rapid and equal distribution of the liquid within the system.

DETAILED DESCRIPTION OF THE INVENTION

I. Overview

[0044] As routinely practiced in modern medicine, sterilized preparations
of concentrated immunoglobulins (especially IgGs) are used for treating
medical conditions that fall into three main classes: immune
deficiencies, inflammatory and autoimmune diseases, and acute infections.
One commonly used IgG product, intravenous immunoglobulin or IVIG, is
formulated for intravenous administration, for example, at a
concentration of at or about 10% IgG. Concentrated immunoglobulins may
also be formulated for subcutaneous or intramuscular administration, for
example, at a concentration at or about 20% IgG. For ease of discussion,
such subcutaneously or intramuscularly formulated IgG compositions are
also included in the term "IVIG" in this application.

[0045] In certain aspects, the present invention provides methods for IVIG
manufacture that increase the final yield of the product, yet still
provide IVIG compositions of equal or higher quality and in some cases
higher concentrations. In one embodiment, the present invention provides
modified Cohn fractionation methods that reduce IgG loss at one or more
precipitation steps.

[0046] In another aspect, the present invention provides IgG compositions
prepared according to the improved manufacturing methods provided herein.
Advantageously, these compositions are less expensive to prepare than
commercial products currently available due to the improved yield
afforded by the methods provided herein. Furthermore, these compositions
are as pure, if not more pure, than compositions manufactured using
commercial methods. Importantly, these compositions are suitable for use
in IVIG therapy for immune deficiencies, inflammatory and autoimmune
diseases, and acute infections. In one embodiment, the IgG composition is
at or about 10% IgG for intravenous administration. In another
embodiment, the IgG composition is at or about 20% for subcutaneous or
intramuscular administration.

[0047] In another aspect, the present invention provides pharmaceutical
compositions and formulations of IgG compositions prepared according to
the improved manufacturing methodologies provided herein. In certain
embodiments, these compositions and formulations provide improved
properties as compared to other IVIG compositions currently on the
market. For example, in certain embodiments, the compositions and
formulations provided herein are stable for an extended period of time.

[0048] In yet another aspect, the present invention provides method for
treating immune deficiencies, inflammatory and autoimmune diseases, and
acute infections comprising the administration of an IgG composition
prepared using the improved methods provided herein.

II. Methods of IVIG Manufacture

[0049] Generally, immunoglobulin preparations according to the present
invention can be prepared from any suitable starting materials, for
example, recovered plasma or source plasma. In a typical example, blood
or plasma is collected from healthy donors. Usually, the blood is
collected from the same species of animal as the subject to which the
immunoglobulin preparation will be administered (typically referred to as
"homologous" immunoglobulins). The immunoglobulins are isolated from the
blood by suitable procedures, such as, for example, precipitation
(alcohol fractionation or polyethylene glycol fractionation),
chromatographic methods (ion exchange chromatography, affinity
chromatography, immunoaffinity chromatography, etc.) ultracentrifugation,
and electrophoretic preparation, and the like. (See, e.g., Cohn et al.,
J. Am. Chem. Soc. 68:459-75 (1946); Oncley et al., J. Am. Chem. Soc.
71:541-50 (1949); Barundern et al., Vox Sang. 7:157-74 (1962); Koblet et
al., Vox Sang. 13:93-102 (1967); U.S. Pat. Nos. 5,122,373 and 5,177,194;
the disclosures of which are hereby incorporated by reference in their
entireties for all purposes).

[0050] In many cases, immunoglobulins are prepared from gamma
globulin-containing products produced by alcohol fractionation and/or ion
exchange and affinity chromatography methods well known to those skilled
in the art. For example, purified Cohn Fraction II is commonly used as a
starting point for the isolation of immunoglobulins. The starting Cohn
Fraction II paste is typically about 95 percent IgG and is comprised of
the four IgG subtypes. The different subtypes are present in Fraction II
in approximately the same ratio as they are found in the pooled human
plasma from which they are obtained. The Fraction II is further purified
before formulation into an administrable product. For example, the
Fraction II paste can be dissolved in a cold purified aqueous alcohol
solution and impurities removed via precipitation and filtration.
Following the final filtration, the immunoglobulin suspension can be
dialyzed or diafiltered (e.g., using ultrafiltration membranes having a
nominal molecular weight limit of less than or equal to 100,000 daltons)
to remove the alcohol. The solution can be concentrated or diluted to
obtain the desired protein concentration and can be further purified by
techniques well known to those skilled in the art.

[0051] Furthermore, additional preparative steps can be used to enrich a
particular isotype or subtype of immunoglobulin. For example, protein A,
protein G or protein H sepharose chromatography can be used to enrich a
mixture of immunoglobulins for IgG, or for specific IgG subtypes. See
generally Harlow and Lane, Using Antibodies, Cold Spring Harbor
Laboratory Press (1999); Harlow and Lane, Antibodies, A Laboratory
Manual, Cold Spring Harbor Laboratory Press (1988); and U.S. Pat. No.
5,180,810, the disclosures of which are hereby incorporated by reference
in their entireties for all purposes.

[0052] Unlike the methods described above, in one aspect the present
invention provides methods of preparing concentrated IgG compositions
that utilize a cryo-poor starting material. Generally, the methods
provided herein utilize both modified Cohn-Oncley alcohol fractionation
steps and ion exchange chromatography to provide superior IgG yields,
while maintaining the same, if not improved, quality as found in
currently available commercial IVIG preparations. For example, in certain
embodiments methods are provided that yield a final bulk IgG composition
containing close to 75% of the IgG content found in the raw plasma
starting material. These methods represent at least a 10% to 12% increase
in the overall IgG yield over existing state of the art purification
methods. For example, it is estimated that the GAMMAGARD® LIQUID
manufacturing process provide a final yield of between about 60% and 65%
of the IgG content found in the starting material. As such, the methods
provided herein provide a significant improvement over the existing IgG
purification technologies.

[0053] In one embodiment, the present invention provides a purified IgG
composition that contains at least 70% of the IgG content found in the
raw plasma starting material. In another embodiment, a purified IgG
composition is provided that contains at least 75% of the IgG content
found in the raw plasma starting material. In other embodiments, a
purified IgG composition provided herein will contain at least about 65%
of the IgG content found in the raw plasma starting material, or at least
66%, 67%, 68%, 69%, 70%, 71%, 72% 73%, 74%, 75%, or more of the IgG
content found in the raw plasma starting material.

[0055] In one aspect, the present invention provides improved methods for
the manufacture of IgG compositions suitable for use in IVIG therapy.
Generally, these methods provide IgG preparations having higher yields
and comparable if not higher purity than current methods employed for the
production of commercial IVIG products.

[0056] In one specific aspect, the present invention provides a method for
preparing a composition of concentrated IgG from plasma, e.g., 10% IVIG,
the method comprising performing at least one alcohol precipitation step
and at least one ion exchange chromatography step. In particular, several
steps in the improved upstream process are different from prior
processes, e.g., the use of 25% ethanol at lower temperatures, ethanol
addition by spraying, pH adjustment by spraying, and the use of finely
divided silica particles.

[0057] In a certain embodiment, the method comprises the steps of (a)
precipitating a cryo-poor plasmid fraction, in a first precipitation
step, with between about 6% and about 10% alcohol at a pH of between
about 6.7 and about 7.3 to obtain a supernatant enriched in IgG, (b)
precipitating IgG from the supernatant with between about 20% and about
30% alcohol at a lower temperature and at a pH of between about 6.7 and
about 7.3 to form a first precipitate, (c) re-suspending the first
precipitate formed in step (b) to form a suspension, (d) treating the
suspension formed in step (c) with a detergent, (e) precipitating IgG
from the suspension with between about 20% and about 30% alcohol at a pH
of between about 6.7 and about 7.3 to form a second precipitate, (f)
re-suspending the second precipitate formed in step (e) to form a
suspension, (g) treating the suspension formed in step (f) with a solvent
and/or detergent, and (h) performing at least one ion exchange
chromatography fractionation thereby preparing a composition of
concentrated IgG. In one embodiment, the method further comprises
treating the suspension formed in step (c) with finely divided silica
dioxide (SiO2) and filtering the solution prior to step (d).

[0058] In one embodiment, a method for preparing a concentrated IgG
composition from plasma is provided, the method comprising the steps of
(a) adjusting the pH of a cryo-poor plasma fraction to about 7.0, (b)
adjusting the ethanol concentration of the cryo-poor plasma fraction of
step (a) to at or about 25% (v/v) at a temperature between about
-5° C. and about -9° C., thereby forming a mixture, wherein
the ethanol concentration may be adjusted by spraying, (c) separating
liquid and precipitate from the mixture of step (b), (d) re-suspending
the precipitate of step (c) with a buffer containing phosphate and
acetate, wherein the pH of the buffer is adjusted with between about 400
and about 700 ml of glacial acetic acid per 1000 L of buffer, thereby
forming a suspension, (e) mixing finely divided silicon dioxide (SiO2)
with the suspension from step (d) for at least about 30 minutes, (f)
filtering the suspension with a filter press, thereby forming a filtrate,
(g) washing the filter press with at least 3 filter press dead volumes of
a buffer containing phosphate and acetate, wherein the pH of the buffer
is adjusted with about 150 ml of glacial acetic acid per 1000 L of
buffer, thereby forming a wash solution, (h) combining the filtrate of
step (f) with the wash solution of step (g), thereby forming a solution,
and treating the solution with a detergent, (i) adjusting the pH of the
solution of step (h) to about 7.0 and adding ethanol to a final
concentration of at or about 25%, thereby forming a precipitate, wherein
the ethanol concentration and/or pH may be adjusted by spraying (j)
separating liquid and precipitate from the mixture of step (i), (k)
dissolving the precipitate in an aqueous solution comprising a solvent or
detergent and maintaining the solution for at least 60 minutes, (l)
passing the solution after step (k) through a cation exchange
chromatography column and eluting proteins absorbed on the column in an
eluate, (m) passing the eluate from step (l) through an anion exchange
chromatography column to generate an effluent (i.e., flow-through), (n)
passing the effluent from step (m) through a nanofilter to generate a
nanofiltrate, (o) passing the nanofiltrate from step (n) through an
ultrafiltration membrane to generate an ultrafiltrate, and (p)
diafiltrating the ultrafiltrate from step (o) against a diafiltration
buffer to generate a diafiltrate having a protein concentration between
about 8% (w/v) and about 22% (w/v), thereby obtaining a composition of
concentrated IgG. In one embodiment, the temperature of step (b) is at or
about -7° C. In one specific embodiment, the suspension buffer in
step (d) is adjusted with about 600 mL glacial acetic acid.

[0059] In certain embodiments, the diafiltrate will have a protein
concentration between about 8% and about 12%, for example, about 8%, or
about 9%, 10%, 11%, or 12%. In a preferred embodiment, the diafiltrate
will have a protein concentration of at or about 10%. In another
preferred embodiment, the diafiltrate will have a protein concentration
of at or about 11%. In yet another preferred embodiment, the diafiltrate
will have a protein concentration of at or about 12%. In other
embodiments, the diafiltrate will have a protein concentration between
about 13% and about 17%, for example, about 13%, or about 14%, 15%, 16%,
or 17%. In yet other embodiments, the diafiltrate will have a protein
concentration between about 18% and about 22%, for example, about 18%, or
about 19%, 20%, 21%, or 22%. In a preferred embodiment, the diafiltrate
will have a protein concentration of at or about 20%. In another
preferred embodiment, the diafiltrate will have a protein concentration
of at or about 21%. In yet another preferred embodiment, the diafiltrate
will have a protein concentration of at or about 22%.

[0060] In certain embodiments of the present invention, the methods
provided herein may comprise improvements in two or more of the
fractionation process steps described above. For example, embodiments may
include improvements in the first precipitation step, the Modified
Fraction II+III precipitation step, the Modified Fraction II+III
dissolution step, and/or the Modified Fraction II+III suspension
filtration step.

[0061] In one embodiment, the improvement made in the first precipitation
step is the addition of alcohol by spraying. In another embodiment, the
improvement made in the first precipitation step is the addition of a pH
modifying agent by spraying. In yet embodiment, the improvement made in
the first precipitation step is the adjustment of the pH of the solution
after addition of the alcohol. In a related embodiment, the improvement
made in the first precipitation step is the maintenance of the pH during
the addition of the alcohol. In another related embodiment, the
improvement made in the first precipitation step is the maintenance of
the pH during the precipitation incubation time by continuously adjusting
the pH of the solution. In certain embodiments, the first precipitation
step may be improved by implementing more than one of these improvements.
Further improvements that may be realized in this step will be evident
from the section provided below discussing the first precipitation
step--Modified Fractionation I. By implementing one or more of the
improvements described above, a reduced amount of IgG is lost in the
precipitate fraction of the first precipitation step and/or a reduced
fraction of IgG is irreversibly denatured during the precipitation step.

[0062] In one embodiment, the improvement made in the Modified Fraction
II+III precipitation step is the addition of alcohol by spraying. In
another embodiment, the improvement made in the Modified Fraction II+III
precipitation step is the addition of a pH modifying agent by spraying.
In yet embodiment, the improvement made in the Modified Fraction II+III
precipitation step is the adjustment of the pH of the solution after
addition of the alcohol. In a related embodiment, the improvement made in
the Modified Fraction II+III precipitation step is the maintenance of the
pH during the addition of the alcohol. In another related embodiment, the
improvement made in the Modified Fraction II+III precipitation step is
the maintenance of the pH during the precipitation incubation time by
continuously adjusting the pH of the solution. In another aspect, the
Modified Fraction II+III precipitation step is improved by increasing the
concentration of alcohol to at or about 25%. In yet another embodiment,
the Modified Fraction II+III precipitation step is improved by lowering
the incubation temperature to between about -7° C. and -9°
C. In certain embodiments, the Modified Fraction II+III precipitation
step may be improved by implementing more than one of these improvements.
Further improvements that may be realized in this step will be evident
from the section provided below discussing the second precipitation
step--Modified Fractionation II+III. By implementing one or more of the
improvements described above, a reduced amount of IgG is lost in the
supernatant fraction of the Modified Fraction II+III precipitation step
and/or a reduced fraction of IgG is irreversibly denatured during the
precipitation step.

[0063] In one embodiment, the improvement made in the Modified Fraction
II+III dissolution step is achieved by increasing the glacial acetic acid
content of the dissolution buffer to about 0.06%. In another embodiment,
the improvement made in the Modified Fraction II+III dissolution step is
achieved by maintaining the pH of the solution during the dissolution
incubation time by continuously adjusting the pH of the solution. In
another embodiment, the improvement made in the Modified Fraction II+III
dissolution step is achieved by mixing finely divided silicon dioxide
(SiO2) with the Fraction II+III suspension prior to filtration. In
certain embodiments, the Modified Fraction II+III dissolution step may be
improved by implementing more than one of these improvements. Further
improvements that may be realized in this step will be evident from the
section provided below discussing the Modified Fraction II+III
dissolution step--Extraction of the Modified Fraction II+III Precipitate.
By implementing one or more of the improvements described above, an
increased amount of IgG is recovered in the Fraction II+III suspension
and/or the amount of impurities is reduced in the Fraction II+III
suspension.

[0064] An exemplary improvement made in the Modified Fraction II+III
suspension filtration step is realized by post-washing the filter with at
least about 3.6 dead volumes of dissolution buffer containing at or about
150 mL glacial acetic acid per 1000 L. Further improvements that may be
realized in this step will be evident from the section provided below
discussing the Modified Fraction II+III suspension filtration
step--Pretreatment and Filtration of the Modified Fraction II+III
Suspension. By implementing one or more of the improvements described
above, a reduced amount of IgG is lost during the Modified Fraction
II+III suspension filtration step.

[0065] In one embodiment, the method may comprise an improvement in the
first precipitation step and the Modified Fraction II+III precipitation
step.

[0066] In another embodiment, the method may comprise an improvement in
the first precipitation step and the Modified Fraction II+III dissolution
step.

[0067] In another embodiment, the method may comprise an improvement in
the first precipitation step and the Modified Fraction II+III suspension
filtration step.

[0068] In another embodiment, the method may comprise an improvement in
the Modified Fraction II+III precipitation step and the Modified Fraction
II+III dissolution step.

[0069] In another embodiment, the method may comprise an improvement in
the Modified Fraction II+III precipitation step and the Modified Fraction
II+III suspension filtration step.

[0070] In another embodiment, the method may comprise an improvement in
the Modified Fraction II+III dissolution step and the Modified Fraction
II+III suspension filtration step.

[0071] In another embodiment, the method may comprise an improvement in
the first precipitation step, the Modified Fraction II+III precipitation
step, and the Modified Fraction II+III dissolution step.

[0072] In another embodiment, the method may comprise an improvement in
the first precipitation step, the Modified Fraction II+III precipitation
step, and the Modified Fraction II+III suspension filtration step.

[0073] In another embodiment, the method may comprise an improvement in
the first precipitation step, the Modified Fraction II+III dissolution
step, and the Modified Fraction II+III suspension filtration step.

[0074] In another embodiment, the method may comprise an improvement in
the Modified Fraction II+III precipitation step, the Modified Fraction
II+III dissolution step, and the Modified Fraction II+III suspension
filtration step.

[0075] In another embodiment, the method may comprise an improvement in
all of the first precipitation step, the Modified Fraction II+III
precipitation step, the Modified Fraction II+III dissolution step, and
the Modified Fraction II+III suspension filtration step.

[0076] In certain embodiments, one process improvement in the IgG
purification methods provided herein comprises the spray addition of one
or more solutions that would otherwise be introduced into a plasma
fraction by fluent addition. For example, in certain embodiments the
process improvement comprises the addition of alcohol (e.g., ethanol)
into a plasma fraction for the purposes of precipitation of one or more
protein species by spraying. In other embodiments, solutions that may be
added to a plasma fraction by spraying include, without limitation, a pH
modifying solution, a solvent solution, a detergent solution, a dilution
buffer, a conductivity modifying solution, and the like. In a preferred
embodiment, one or more alcohol precipitation steps is performed by the
addition of alcohol to a plasma fraction by spraying. In a second
preferred embodiment, one or more pH adjustment steps is performed by the
addition of a pH modifying solution to a plasma fraction by spraying.

[0077] In certain embodiments, another process improvement, which may be
combined with any other process improvement, comprises the adjustment of
the pH of a plasma fraction being precipitated after and/or concomitant
with the addition of the precipitating agent (e.g., alcohol or
polyethelene glycol). In some embodiments, a process improvement is
provided in which the pH of a plasma fraction being actively precipitated
is maintained throughout the entire precipitation incubation or hold step
by continuous monitoring and adjustment of the pH. In preferred
embodiments the adjustment of the pH is performed by the spray addition
of a pH modifying solution.

[0078] In other embodiments, another process improvement, which may be
combined with any other process improvement, comprises the use of a
finely divided silica treatment step to remove impurities.

[0079] 1. Preparation of Cryo-Poor Plasma

[0080] The starting material used for the preparation of concentrated IgG
compositions generally consists of either recovered plasma (i.e., plasma
that has been separated from whole blood ex vivo) or source plasma (i.e.,
plasma collected via plasmapheresis). The purification process typically
starts with thawing previously frozen pooled plasma, which has already
been assayed for safety and quality considerations. Thawing is typically
carried out at a temperature no higher than 6° C. After complete
thawing of the frozen plasma at low temperature, centrifugation is
performed in the cold (e.g., ≦6° C.) to separate solid
cryo-precipitates from the liquid supernatant. Alternatively, the
separation step can be performed by filtration rather than
centrifugation. The liquid supernatant (also referred to as "cryo-poor
plasma," after cold-insoluble proteins removed by centrifugation from
fresh thawed plasma) is then processed in the next step. Various
additional steps can be taken at this juncture for the isolation of
factor eight inhibitor bypass activity (FEIBA), Factor IX-complex, Factor
VII-concentrate, or Antithrombin III-complex.

[0081] 2. First Precipitation Event--Modified Fractionation I

[0082] In this step, cryo-poor plasma is typically cooled to about
0±1° C. and the pH is adjusted to between about 7.0 and about
7.5, preferably between about 7.1 and about 7.3, most preferably about
7.2. In one embodiment, the pH of the cryo-poor plasma is adjusted to a
pH of at or about 7.2. Pre-cooled ethanol is then added while the plasma
is stirred to a target concentration of ethanol at or about 8% v/v. At
the same time the temperature is further lowered to between about -4 and
about 0° C. In a preferred embodiment, the temperature is lowered
to at or about -2° C., to precipitate contaminants such as
α2-macroglobulin, β1A- and β1C-globulin,
fibrinogen, and Factor VIII. Typically, the precipitation event will
include a hold time of at least about 1 hour, although shorter or longer
hold times may also be employed. Subsequently, the supernatant
(Supernatant I), ideally containing the entirety of the IgG content
present in the cryo-poor plasma, is then collected by centrifugation,
filtration, or another suitable method.

[0083] As compared to conventional methods employed as a first
fractionation step for cryo-poor plasma (Cohn et al., supra; Oncley et
al., supra), the present invention provides, in several embodiments,
methods that result in improved IgG yields in the Supernatant I fraction.
In one embodiment, the improved IgG yield is achieved by adding the
alcohol by spraying. In another embodiment, the improved IgG yield is
achieved by adding a pH modifying agent by spraying. In yet another
embodiment, the improved IgG yield is achieved by adjusting the pH of the
solution after addition of the alcohol. In a related embodiment, the
improved IgG yield is achieved by adjusting the pH of the solution during
the addition of the alcohol.

[0084] In one specific aspect, the improvement relates to a method in
which a reduced amount of IgG is lost in the precipitate fraction of the
first precipitation step. For example, in certain embodiments, a reduced
amount of IgG is lost in the precipitate fraction of the first
precipitation step as compared to the amount of IgG lost in the first
precipitation step of the Cohn method 6 protocol.

[0085] In certain embodiments, the process improvement is realized by
adjusting the pH of the solution to between about 7.0 and about 7.5 after
the addition of the precipitating alcohol. In other embodiments, the pH
of the solution is adjusted to between about 7.1 and about 7.3 after
addition of the precipitating alcohol. In yet other embodiments, the pH
of the solution is adjusted to about 7.0 or about 7.1, 7.2, 7.3, 7.4, or
7.5 after addition of the precipitating alcohol. In a particular
embodiment, the pH of the solution is adjusted to about 7.2 after
addition of the precipitating alcohol. As such, in certain embodiments, a
reduced amount of IgG is lost in the precipitate fraction of the first
precipitation step as compared to an analogous precipitation step in
which the pH of the solution is adjusted prior to but not after addition
of the precipitating alcohol. In one embodiment, the pH is maintained at
the desired pH during the precipitation hold or incubation time by
continuously adjusting the pH of the solution. In one embodiment, the
alcohol is ethanol.

[0086] In other certain embodiments, the process improvement is realized
by adding the precipitating alcohol and/or the solution used to adjust
the pH by spraying, rather than by fluent addition. As such, in certain
embodiments, a reduced amount of IgG is lost in the precipitate fraction
of the first precipitation step as compared to an analogous precipitation
step in which the alcohol and/or solution used to adjust the pH is
introduced by fluent addition. In one embodiment, the alcohol is ethanol.

[0087] In yet other certain embodiments, the improvement is realized by
adjusting the pH of the solution to between about 7.0 and about 7.5. In a
preferred embodiment, the pH of the solution is adjusted to between about
7.1 and about 7.3. In other embodiments, the pH of the solution is
adjusted to at or about 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5 after the
addition of the precipitating alcohol and by adding the precipitating
alcohol and/or the solution used to adjust the pH by spraying, rather
than by fluent addition. In a particular embodiment, the pH of the
solution is adjusted to at or about 7.2 after addition of the
precipitating alcohol and by adding the precipitating alcohol and/or the
solution used to adjust the pH by spraying, rather than by fluent
addition. In one embodiment, the alcohol is ethanol.

[0088] 3. Second Precipitation Event--Modified Fractionation II+III

[0089] To further enrich the IgG content and purity of the fractionation,
Supernatant I is subjected to a second precipitation step, which is a
modified Cohn-Oncley Fraction II+III fractionation. Generally, the pH of
the solution is adjusted to a pH of between about 6.6 and about 6.8. In a
preferred embodiment, the pH of the solution is adjusted to at or about
6.7. Alcohol, preferably ethanol, is then added to the solution while
being stirred to a final concentration of between about 20% and about 25%
(v/v) to precipitate the IgG in the fraction. In a preferred embodiment,
alcohol is added to a final concentration of at or about 25% (v/v) to
precipitate the IgG in the fraction. Generally, contaminants such as
α1-lipoprotein, α1-antitrypsin, Gc-globulins,
α1X-glycoprotin, haptoglobulin, ceruloplasmin, transferrin,
hemopexin, a fraction of the Christmas factor, thyroxin binding globulin,
cholinesterase, hypertensinogen, and albumin will not be precipitated by
these conditions.

[0090] Prior to or concomitant with alcohol addition, the solution is
further cooled to between about -7° C. and about -9° C. In
a preferred embodiment, the solution is cooled to a temperature at or
about -7° C. After completion of the alcohol addition, the pH of
the solution is immediately adjusted to between about 6.8 and about 7.0.
In a preferred embodiment, the pH of the solution is adjusted to at or
about 6.9. Typically, the precipitation event will include a hold time of
at least about 10 hours, although shorter or longer hold times may also
be employed. Subsequently, the precipitate (Modified Fraction II+III),
which ideally contains at least about 85%, preferably at least about 90%,
more preferably at least about 95%, of the IgG content present in the
cryo-poor plasma, is separated from the supernatant by centrifugation,
filtration, or another suitable method and collected. As compared to
conventional methods employed as a second fractionation step for
cryo-poor plasma (Cohn et al., supra; Oncley et al., supra), the present
invention provides, in several embodiments, methods that result in
improved IgG yields in the Modified Fraction II+III precipitate. In a
related embodiment, the present invention provides methods that result in
a reduced loss of IgG in the Modified II+III supernatant.

[0091] As compared to conventional methods employed as a second
fractionation step for cryo-poor plasma (Cohn et al., supra; Oncley et
al., supra), the present invention provides, in several embodiments,
methods that result in improved IgG yields in the Modified Fraction
II+III precipitate. In one embodiment, the improvement is realized by the
addition of alcohol by spraying. In another embodiment, the improvement
is realized by the addition of a pH modifying agent by spraying. In
another embodiment, the improvement is realized by adjusting the pH of
the solution after addition of the alcohol. In a related embodiment, the
improvement is realized by adjusting the pH of the solution during
addition of the alcohol. In another embodiment, the improvement is
realized by increasing the concentration of alcohol (e.g., ethanol) to
about 25% (v/v). In another embodiment, the improvement is realized by
lowering the temperature of the precipitation step to between about
-7° C. and -9° C. In a preferred embodiment, the
improvement is realized by increasing the concentration of alcohol (e.g.,
ethanol) to about 25% (v/v) and lowing the temperature to between about
-7° C. and -9° C. In comparison, both Cohn et al. and
Oncley et al. perform precipitation at -5° C. and Oncley et al.
use 20% alcohol, in order to reduce the level of contaminants in the
precipitate. Advantageously, the methods provided herein allow for
maximal IgG yield without high levels of contamination in the final
product.

[0092] It has been discovered that when the pH of the solution is adjusted
to a pH of about 6.9 prior to addition of the precipitating alcohol, the
pH of the solution shift from 6.9 to between about 7.4 and about 7.7, due
in part to protein precipitation (see, FIG. 8). As the pH of the solution
shifts away from 6.9, precipitation of IgG becomes less favorable and the
precipitation of certain contaminants becomes more favorable.
Advantageously, the inventors have found that by adjusting the pH of the
solution after addition of the precipitating alcohol, that a higher
percentage of IgG is recovered in the Fraction II+III precipitate.

[0093] Accordingly, in one aspect, the improvement relates to a method in
which a reduced amount of IgG is lost in the supernatant fraction of the
modified Fraction II+III precipitation step. In other words, an increased
percentage of the starting IgG is present in the Fraction II+III
precipitate. In certain embodiments, the process improvement is realized
by adjusting the pH of the solution to between about 6.7 and about 7.1
immediately after or during the addition of the precipitating alcohol. In
another embodiment, the process improvement is realized by maintaining
the pH of the solution to between about 6.7 and about 7.1 continuously
during the precipitation incubation period. In other embodiments, the pH
of the solution is adjusted to between about 6.8 and about 7.0
immediately after or during the addition of the precipitating alcohol, or
to a pH of about 6.7, 6.8, 6.9, 7.0, or 7.1 immediately after or during
the addition of the precipitating alcohol. In a particular embodiment,
the pH of the solution is adjusted to about 6.9 immediately after or
during the addition of the precipitating alcohol. In certain embodiments,
the pH of the solution is maintained at between about 6.8 to about 7.0
continuously during the precipitation incubation period, or at a pH of
about 6.9 continuously during the precipitation incubation period. As
such, in certain embodiments, a reduced amount of IgG is lost in the
supernatant fraction of the second precipitation step as compared to an
analogous precipitation step in which the pH of the solution is adjusted
prior to but not after addition of the precipitating alcohol or to an
analogous precipitation step in which the pH of the solution is not
maintained during the entirety of the precipitation incubation period. In
one embodiment, the pH is maintained at the desired pH during the
precipitation hold or incubation time by continuously adjusting the pH of
the solution. In one embodiment, the alcohol is ethanol.

[0094] In another embodiment, the process improvement is realized by
adding the precipitating alcohol and/or the solution used to adjust the
pH by spraying, rather than by fluent addition. As such, in certain
embodiments, a reduced amount of IgG is lost in the supernatant fraction
of the second precipitation step as compared to an analogous
precipitation step in which the alcohol and/or solution used to adjust
the pH is introduced by fluent addition. In one embodiment, the alcohol
is ethanol.

[0095] In another embodiment, the process improvement is realized by
performing the precipitation step at a temperature between about
-7° C. and about -9° C. In one embodiment, the
precipitation step is performed at a temperature of at or about
-7° C. In another embodiment, the precipitation step is performed
at a temperature of at or about -8° C. In another embodiment, the
precipitation step is performed at a temperature of at or about
-9° C. In certain embodiments, the alcohol concentration of the
precipitation step is between about 23% and about 27%. In a preferred
embodiment, the alcohol concentration is between about 24% and about 26%.
In another preferred embodiment, the alcohol concentration is at or about
25%. In other embodiments, the alcohol concentration may be at or about
23%, 24%, 25%, 26%, or 27%. In a particular embodiment, the second
precipitation step is performed at a temperature of at or about
-7° C. with an alcohol concentration of at or about 25%. In one
embodiment, the alcohol is ethanol.

[0096] The effect of increasing the alcohol concentration of the second
precipitation from 20%, as used in Oncley et al., supra, to 25% and
lowering the temperature of the incubation from -5° C., as used in
the Cohn and Oncley methods, to at or about -7° C. is a 5% to 6%
increase in the IgG content of the modified Fraction II+III precipitate.

[0097] In another embodiment, the process improvement is realized by
adjusting the pH of the solution to between about 6.7 and about 7.1,
preferably at or about 6.9, immediately after or during the addition of
the precipitating alcohol, maintaining the pH of the solution at a pH of
between about 6.7 and about 7.1, preferably at or about 6.9, by
continuously adjusting the pH during the precipitation incubation period,
and by adding the precipitating alcohol and/or the solution used to
adjust the pH by spraying, rather than by fluent addition. In another
particular embodiment, the process improvement is realized by performing
the precipitation step at a temperature between about -7° C. and
about -9° C., preferably at or about -7° C. and by
precipitating the IgG with an alcohol concentration of between about 23%
and about 27%, preferably at or about 25%. In yet another particular
embodiment, the process improvement is realized by incorporating all of
the Modified Fraction II+III improvements provided above. In a preferred
embodiment, the process improvement is realized by precipitating IgG at a
temperature of at or about -7° C. with at or about 25% ethanol
added by spraying and then adjusting the pH of the solution to at or
about 6.9 after addition of the precipitating alcohol. In yet another
preferred embodiment, the pH of the solution is maintained at or about
6.9 for the entirety of the precipitation incubation or hold time.

[0098] 4. Extraction of the Modified Fraction II+III Precipitate

[0099] In order to solubilize the IgG content of the modified Fraction
II+III precipitate, a cold extraction buffer is used to re-suspend the
Fractionation II+III precipitate at a typical ratio of 1 part precipitate
to 15 parts of extraction buffer. Other suitable re-suspension ratios may
be used, for example from about 1:8 to about 1:30, or from about 1:10 to
about 1:20, or from about 1:12 to about 1:18, or from about 1:13 to about
1:17, or from about 1:14 to about 1:16. In certain embodiments, the
re-suspension ratio may be about 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14,
1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26,
1:27, 1:28, 1:29, 1:30, or higher.

[0100] Suitable solutions for the extraction of the modified II+III
precipitate will generally have a pH between about 4.0 and about 5.5. In
certain embodiments, the solution will have a pH between about 4.5 and
about 5.0, in other embodiments, the extraction solution will have a pH
of about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2,
5.3, 5.4, or 5.5. In a preferred embodiment, the pH of the extraction
buffer will be at or about 4.5. In another preferred embodiment, the pH
of the extraction buffer will be at or about 4.7. In another preferred
embodiment, the pH of the extraction buffer will be at or about 4.9.
Generally, these pH requirements can be met using a buffering agent
selected from, for example, acetate, citrate, monobasic phosphate,
dibasic phosphate, mixtures thereof, and the like. Suitable buffer
concentrations typically range from about 5 to about 100 mM, or from
about 10 to about 50 mM, or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,
55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mM buffering agent.

[0101] The extraction buffer will preferably have a conductivity of from
about 0.5 mScm-1 to about 2.0 mScm-1. For example, in certain
embodiments, the conductivity of the extraction buffer will be about 0.5
mScm-1, or about 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,
1.6, 1.7, 1.8, 1.9, or about 2.0 mScm-1. One of ordinary skill in
the art will know how to generate extraction buffers having an
appropriate conductivity.

[0102] In one particular embodiment, an exemplary extraction buffer may
contain at or about 5 mM monobasic sodium phosphate and at or about 5 mM
acetate at a pH of at or about 4.5±0.2 and conductivity of at or about
0.7 to 0.9 mS/cm.

[0103] Generally, the extraction is performed at between about 0°
C. and about 10° C., or between about 2° C. and about
8° C. In certain embodiments, the extraction may be performed at
about 0° C., 1° C., 2° C., 3° C., 4°
C., 5° C., 6° C., 7° C., 8° C., 9° C.,
or 10° C. In a particular embodiment, the extraction is performed
at between about 2° C. and about 10° C. Typically, the
extraction process will proceed for between about 60 and about 300
minutes, or for between about 120 and 240 min, or for between about 150
and 210 minutes, while the suspension is continuously stirred. In certain
embodiments, the extraction process will proceed for about 60, 70, 80,
90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,
240, 250, 260, 270, 280, 290, or about 300 minutes. In a preferred
embodiment, the extraction process will proceed for at least 160 minutes
with continuous stifling.

[0104] It has been found that employing an extraction buffer containing 5
mM monobasic sodium phosphate, 5 mM acetate, and 0.051% to 0.06% glacial
acetic acid (v/v), a substantial increase in the yield increase in the
final IgG composition can be obtained without jeopardizing the purity of
the final product. The correlation of amount of acetic acid and
extraction buffer pH is demonstrated in FIG. 9. In a preferred
embodiment, the Fraction II+III precipitate is extracted with a paste to
buffer ration of at or about 1:15 at a pH of at or about 4.5±0.2.

[0105] Advantageously, it has been found that compared to the current
manufacturing process for GAMMAGARD® LIQUID (Baxter Healthcare),
which employs an extraction buffer containing 5 mM monobasic sodium
phosphate, 5 mM acetate, and 0.051% glacial acetic acid (v/v), that by
increasing the glacial acetic acid content to at or about 0.06% (v/v), a
substantial increase in the yield increase in the final IgG composition
can be obtained. As compared to methods previously employed for the
extraction of the precipitate formed by the second precipitation step
(GAMMAGARD® LIQUID), the present invention provides, in several
embodiments, methods that result in improved IgG yields in the Modified
Fraction II+III suspension.

[0106] In one aspect, the improvement relates to a method in which a
reduced amount of IgG is lost in the non-solubilized fraction of the
Modified Fraction II+III precipitate. In one embodiment, the process
improvement is realized by extracting the Modified Fraction II+III
precipitate at a ratio of 1:15 (precipitate to buffer) with a solution
containing 5 mM monobasic sodium phosphate, 5 mM acetate, and 0.06%
glacial acetic acid (v/v). In another embodiment, the improvement is
realized by maintaining the pH of the solution during the duration of the
extraction process. In one embodiment, the pH of the solution is
maintained at between about 4.1 and about 4.9 for the duration of the
extraction process. In a preferred embodiment, the pH of the solution is
maintained at between about 4.2 and about 4.8 for the duration of the
extraction process. In a more preferred embodiment, the pH of the
solution is maintained at between about 4.3 and about 4.7 for the
duration of the extraction process. In another preferred embodiment, the
pH of the solution is maintained at between about 4.4 and about 4.6 for
the duration of the extraction process. In yet another preferred
embodiment, the pH of the solution is maintained at or at about 4.5 for
the duration of the extraction process.

[0107] In another aspect, the improvement relates to a method in which an
increased amount of IgG is solubilized from the Fraction II+III
precipitate in the Fraction II+III dissolution step. In one embodiment,
the process improvement is realized by solubilizing the Fraction II+III
precipitate in a dissolution buffer containing 600 mL glacial acetic acid
per 1000 L. In another embodiment, the improvement relates to a method in
which impurities are reduced after the IgG in the Fraction II+III
precipitate is solubilized. In one embodiment, the process improvement is
realized by mixing finely divided silicon dioxide (SiO2) with the
Fraction II+III suspension for at least about 30 minutes.

[0109] In order to remove the non-solubilized fraction of the Modified
Fraction II+III precipitate (i.e., the Modified Fraction II+III filter
cake), the suspension is filtered, typically using depth filtration.
Depth filters that may be employed in the methods provided herein
include, metallic, glass, ceramic, organic (such as diatomaceous earth)
depth filters, and the like. Example of suitable filters include, without
limitation, Cuno 50S A, Cuno 90S A, and Cuno VR06 filters (Cuno).
Alternatively, the separation step can be performed by centrifugation
rather than filtration.

[0110] Although the manufacturing process improvements described above
minimize IgG losses in the initial steps of the purification process,
critical impurities, including PKA activity, amidolytic activity, and
fibrinogen content, are much higher when, for example, the II+III paste
is extracted at pH 4.5 or 4.6, as compared to when the extraction occurs
at a pH around 4.9 to 5.0 (see, Examples 2 to 5).

[0111] In order to counter act the impurities extracted in the methods
provided herein, it has now been found that the purity of the IgG
composition can be greatly enhanced by the addition of a pretreatment
step prior to filtration/centrifugation. In one embodiment, this
pretreatment step comprises addition of finely divided silica dioxide
particles (e.g., fumed silica, Aerosil® followed by a 40 to 80 minute
incubation period during which the suspension is constantly mixed. In
certain embodiments, the incubation period will be between about 50
minutes and about 70 minutes, or about 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, or more minutes. Generally, the treatment will be performed
at between about 0° C. and about 10° C., or between about
2° C. and about 8° C. In certain embodiments, the treatment
may be performed at about 0° C., 1° C., 2° C.,
3° C., 4° C., 5° C., 6° C., 7° C.,
8° C., 9° C., or 10° C. In a particular embodiment,
the treatment is performed at between about 2° C. and about
10° C.

[0112] The effect of the fumed silica treatment is exemplified by the
results found in Example 17. In this example, a Fraction II+III
precipitate is suspended and split into two samples, one of which is
clarified with filter aid only prior to filtration (FIG. 7A) and one of
which is treated with fumed silica prior to addition of the filter aid
and filtration (FIG. 7B). As can be seen in the chromatographs and in the
quantitated data, the filtrate sample pretreated with fumed silica had a
much higher IgG purity than the sample only treated with filter aid
(68.8% vs. 55.7%; compare Tables 17 and 18, respectively).

[0113] In certain embodiments, fumed silica is added at a concentration of
between about 20 g/kg II+III paste and about 100 g/kg II+III paste (i.e.,
for a Modified Fraction II+III precipitate that is extracted at a ration
of 1:15, fumed silica should be added at a concentration from about 20
g/16 kg II+III suspension to about 100 g/16 kg II+III suspension, or at a
final concentration of about 0.125% (w/w) to about 0.625% (w/w)). In
certain embodiments, the fumed silica may be added at a concentration of
about 20 g/kg II+III paste, or about 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95, or 100 g/kg II+III paste. In one specific
embodiment, fumed silica (e.g., Aerosil 380 or equivalent) is added to
the Modified Fraction II+III suspension to a final concentration of about
40 g/16 kg II+III. Mixing takes place at about 2 to 8° C. for at
least 50 to 70 minutes.

[0114] In certain embodiments, filter aid, for example Celpure C300
(Celpure) or Hyflo-Supper-Cel (World Minerals), will be added after the
silica dioxide treatment, to facilitate depth filtration. Filter aid can
be added at a final concentration of from about 0.1 kg/kg II+III paste to
about 0.07 kg/kg II+III paste, or from about 0.2 kg/kg II+III paste to
about 0.06 kg/kg II+III paste, or from about 0.3 kg/kg II+III paste to
about 0.05 kg/kg II+III paste. In certain embodiments, the filter aid
will be added at a final concentration of about 0.1 kg/kg II+III paste,
or about 0.2, 0.3, 0.4, 0.5, 0.6, or 0.7 kg/kg II+III paste.

[0115] A significant fraction of IgG was being lost during the filtration
step of the GAMMAGARD® LIQUID manufacturing process. It was found
that the current methods of post-filtration wash, using 1.8 dead volumes
of suspension buffer to purge the filter press frames and lines, were
insufficient for maximal recovery of IgG at this step. Surprisingly, it
was found that at least 3.0 dead volumes, preferably 3.6 dead volumes, of
suspension buffer were required in order for efficient recovery of total
IgG in the Modified Fraction II+III clarified suspension (see, Example 12
and FIG. 1). In certain embodiments, the filter press may be washed with
any suitable suspension buffer. In a particular embodiment, the wash
buffer will comprise, for example, 5 mM monobasic sodium phosphate, 5 mM
acetate, and 0.015% glacial acetic acid (v/v).

[0116] In one aspect, the improvement relates to a method in which a
reduced amount of IgG is lost during the Fraction II+III suspension
filtration step. In one embodiment, the process improvement is realized
by post-washing the filter with at least about 3.6 dead volumes of
dissolution buffer containing 150 mL glacial acetic acid per 1000 L. The
relationship between the amount of glacial acetic acid and pH in the
post-wash buffer is shown in FIG. 10. In one embodiment, the pH of the
post-wash extraction buffer is between about 4.6 and about 5.3. In a
preferred embodiment, the pH of the post-wash buffer is between about 4.7
and about 5.2. In another preferred embodiment, the pH of the post-wash
buffer is between about 4.8 and about 5.1. In yet another preferred
embodiment, the pH of the post-wash buffer is between about 4.9 and about
5.0.

[0117] As compared to methods previously employed for the clarification of
the suspension formed from the second precipitation step (GAMMAGARD®
LIQUID), the present invention provides, in several embodiments, methods
that result in improved IgG yields and purity in the clarified Fraction
II+III suspension. In one aspect, the improvement relates to a method in
which a reduced amount of IgG is lost in the Modified Fraction II+III
filter cake. In other aspect, the improvement relates to a method in
which a reduced amount of an impurity is found in the clarified Fraction
II+III suspension.

[0118] In one embodiment, the process improvements are realized by
inclusion of a fumed silica treatment prior to filtration or centrifugal
clarification of the Modified Fraction II+III suspension. In certain
embodiments, the fumed silica treatment will include addition of from
about 0.1 kg/kg II+III paste to about 0.07 kg/kg II+III paste, or from
about 0.2 kg/kg II+III paste to about 0.06 kg/kg II+III paste, or from
about 0.3 kg/kg II+III paste to about 0.05 kg/kg II+III paste, or about
0.2, 0.3, 0.4, 0.5, 0.6, or 0.7 kg/kg II+III paste, and the mixture will
be incubated for between about 50 minutes and about 70 minutes, or about
30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more minutes at a
temperature between about 2° C. and about 8° C. In another
embodiment, the process improvements are realized by inclusion of a fumed
silica treatment which reduced the levels of residual fibrinogen,
amidolytic activity, and/or prekallikrein activator activity.

[0119] In another embodiment, the process improvements are realized by
washing the depth filter with between about 3 and about 5 volumes of the
filter dead volume after completing the Modified Fraction II+III
suspension filtration step. In certain embodiments, the filter will be
washed with between about 3.5 volumes and about 4.5 volumes, or at least
about 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,
3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 volumes
of the filter dead volume. In a particular embodiment, the filter press
will be washed with at least about 3.6 dead volumes of suspension buffer.

[0120] 6. Detergent Treatment

[0121] In order to remove additional contaminants from the Modified
Fraction II+III filtrate, the sample is next subjected to a detergent
treatment. Methods for the detergent treatment of plasma derived
fractions are well known in the art. Generally, any standard non-ionic
detergent treatment may be used in conjunction with the methods provided
herein. For example, an exemplary protocol for a detergent treatment is
provided below.

[0122] Briefly, polysorbate-80 is added to the Modified Fraction II+III
filtrate at a final concentration of about 0.2% (w/v) with stirring and
the sample is incubated for at least 30 minutes at a temperature between
about 2 to 8° C. Sodium citrate dehydrate is then mixed into the
solution at a final concentration of about 8 g/L and the sample is
incubated for an additional 30 minutes, with continuous of stirring at a
temperature between about 2 to 8° C.

[0124] In one embodiment, a process improvement is realized by adding the
detergent reagents (e.g., polysorbate-80 and sodium citrate dehydrate) by
spraying rather than by fluent addition. In other embodiments, the
detergent reagents may be added as solids to the Modified Fraction II+III
filtrate while the sample is being mixed to ensure rapid distribution of
the additives. In certain embodiments, it is preferable to add solid
reagents by sprinkling the solids over a delocalized surface area of the
filtrate such that local overconcentration does not occur, such as in
fluent addition.

[0125] 7. Third Precipitation Event--Precipitation G

[0126] In order to remove several residual small proteins, such as albumin
and transferrin, a third precipitation is performed at a concentration of
25% alcohol. Briefly, the pH of the detergent treated II+III filtrate is
adjusted to between about 6.8 and 7.2, preferably between about 6.9 and
about 7.1, most preferably about 7.0 with a suitable pH modifying
solution (e.g., 1M sodium hydroxide or 1M acetic acid). Cold alcohol is
then added to the solution to a final concentration of about 25% (v/v)
and the mixture is incubated while stirring at between about -6°
C. to about -10° C. for at least 1 hour to form a third
precipitate (i.e., precipitate G). In one embodiment, the mixture is
incubated for at lease 2 hours, or at least 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more hours. In a
preferred embodiment, the mixture is incubated for at least 2 hours. In a
more preferred embodiment, the mixture is incubated for at least 4 hours.
In an even more preferred embodiment, the mixture is incubated for at
least 8 hours.

[0127] In one aspect, a process improvement relates to a method in which a
reduced amount of IgG is lost in the supernatant fraction of the third
precipitation step. In certain embodiments, the process improvement is
realized by adjusting the pH of the solution to between about 6.8 and
about 7.2 immediately after or during the addition of the precipitating
alcohol. In another embodiment, the process improvement is realized by
maintaining the pH of the solution to between about 6.8 and about 7.2
continuously during the precipitation incubation period. In other
embodiments, the pH of the solution is adjusted to between about 6.9 and
about 7.1 immediately after or during the addition of the precipitating
alcohol, or to a pH of about 6.8, 6.9, 7.0, 7.1, or 7.2 immediately after
or during the addition of the precipitating alcohol. In a particular
embodiment, the pH of the solution is adjusted to about 7.0 immediately
after or during the addition of the precipitating alcohol. In certain
embodiments, the pH of the solution is maintained at between about 6.9 to
about 7.1 continuously during the precipitation incubation period, or at
a pH of about 7.0 continuously during the precipitation incubation
period. As such, in certain embodiments, a reduced amount of IgG is lost
in the supernatant fraction of the third precipitation step as compared
to an analogous precipitation step in which the pH of the solution is
adjusted prior to but not after addition of the precipitating alcohol or
to an analogous precipitation step in which the pH of the solution is not
maintained during the entirety of the precipitation incubation period. In
one embodiment, the pH is maintained at the desired pH during the
precipitation hold or incubation time by continuously adjusting the pH of
the solution. In one embodiment, the alcohol is ethanol.

[0128] In another embodiment, the process improvement is realized by
adding the precipitating alcohol and/or the solution used to adjust the
pH by spraying, rather than by fluent addition. As such, in certain
embodiments, a reduced amount of IgG is lost in the supernatant fraction
of the third precipitation step as compared to an analogous precipitation
step in which the alcohol and/or solution used to adjust the pH is
introduced by fluent addition. In one embodiment, the alcohol is ethanol.

[0129] 8. Suspension and Filtration of Precipitate G (PptG)

[0130] In order to solubilize the IgG content of the precipitate G, a cold
extraction buffer is used to re-suspend the PptG. Briefly, the
precipitate G is dissolved 1 to 3.5 in Water for Injection (WFI) at
between about 0° C. and about 8° C. to achieve an
AU280-320 value of between about 40 to 95. The final pH of the
solution, which is stirred for at least 2 hours, is then adjusted to at
or about 5.2±0.2. In one embodiment, this pH adjustment is performed
with 1M acetic acid. To increase the solubility of IgG, the conductivity
of the suspension is increased to between about 2.5 and about 6.0 mS/cm.
In one embodiment, the conductivity is increased by the addition of
sodium chloride. The suspended PptG solution is then filtered with a
suitable depth filter having a nominal pore size of between about 0.1
μm and about 0.4 μm in order to remove any undissolved particles.
In one embodiment, the nominal pore size of the depth filter is about 0.2
μm (e.g., Cuno VR06 filter or equivalent) to obtain a clarified
filtrate. In another embodiment, the suspended PptG solution is
centrifuged to recover a clarified supernatant. Post-wash of the filter
is performed using a sodium chloride solution with a conductivity of
between about 2.5 and about 6.0 mS/cm. Typically, suitable solutions for
the extraction of precipitate G include, WFI and low conductivity
buffers. In one embodiment, a low conductivity buffer has a conductivity
of less than about 10 mS/cm. In a preferred embodiment, the low
conductivity buffer has a conductivity of less than about 9, 8, 7, 6, 5,
4, 3, 2, or 1 mS/cm. In a preferred embodiment, the low conductivity
buffer has a conductivity of less than about 6 mS/cm. In another
preferred embodiment, the low conductivity buffer has a conductivity of
less than about 4 mS/cm. In another preferred embodiment, the low
conductivity buffer has a conductivity of less than about 2 mS/cm.

[0131] 9. Solvent Detergent Treatment

[0132] In order to inactivate various viral contaminants which may be
present in plasma-derived products, the clarified PptG filtrate is next
subjected to a solvent detergent (S/D) treatment. Methods for the
detergent treatment of plasma derived fractions are well known in the art
(for review see, Pelletier J P et al., Best Pract Res Clin Haematol.
2006; 19(1):205-42). Generally, any standard S/D treatment may be used in
conjunction with the methods provided herein. For example, an exemplary
protocol for an S/D treatment is provided below.

[0133] Briefly, Triton X-100, Tween-20, and tri(n-butyl)phosphate (TNBP)
are added to the clarified PptG filtrate at final concentrations of about
1.0%, 0.3%, and 0.3%, respectively. The mixture is then stirred at a
temperature between about 18° C. and about 25° C. for at
least about an hour.

[0134] In one embodiment, a process improvement is realized by adding the
S/D reagents (e.g., Triton X-100, Tween-20, and TNBP) by spraying rather
than by fluent addition. In other embodiments, the detergent reagents may
be added as solids to the clarified PptG filtrate, which is being mixed
to ensure rapid distribution of the S/D components. In certain
embodiments, it is preferable to add solid reagents by sprinkling the
solids over a delocalized surface area of the filtrate such that local
overconcentration does not occur, such as in fluent addition.

[0135] 10. Ion Exchange Chromatography

[0136] In order to further purify and concentrate IgG from the S/D treated
PptG filtrate, cation exchange and/or anion exchange chromatography can
be employed. Methods for purifying and concentrating IgG using ion
exchange chromatography are well known in the art. For example, U.S. Pat.
No. 5,886,154 describes a method in which a Fraction II+III precipitate
is extracted at low pH (between about 3.8 and 4.5), followed by
precipitation of IgG using caprylic acid, and finally implementation of
two anion exchange chromatography steps. U.S. Pat. No. 6,069,236
describes a chromatographic IgG purification scheme that does not rely on
alcohol precipitation at all. PCT Publication No. WO 2005/073252
describes an IgG purification method involving the extraction of a
Fraction II+III precipitate, caprylic acid treatment, PEG treatment, and
a single anion exchange chromatography step. U.S. Pat. No. 7,186,410
describes an IgG purification method involving the extraction of either a
Fraction I+II+III or a Fraction II precipitate followed by a single anion
exchange step performed at an alkaline pH. U.S. Pat. No. 7,553,938
describes a method involving the extraction of either a Fraction I+II+III
or a Fraction II+III precipitate, caprylate treatment, and either one or
two anion exchange chromatography steps. U.S. Pat. No. 6,093,324
describes a purification method comprising the use of a macroporous anion
exchange resin operated at a pH between about 6.0 and about 6.6. U.S.
Pat. No. 6,835,379 describes a purification method that relies on cation
exchange chromatography in the absence of alcohol fractionation. The
disclosures of the above publications are hereby incorporated by
reference in their entireties for all purposes

[0137] In one embodiment of the methods of the present invention, the S/D
treated PptG filtrate may be subjected to both cation exchange
chromatography and anion exchange chromatography. For example, in one
embodiment, the S/D treated PptG filtrate is passed through a cation
exchange column, which binds the IgG in the solution. The S/D reagents
can then be washed away from the absorbed IgG, which is subsequently
eluted off of the column with a high pH elution buffer having a pH
between about 8.0 and 9.0. In this fashion, the cation exchange
chromatography step can be used to remove the S/D reagents from the
preparation, concentrate the IgG containing solution, or both. In certain
embodiments, the pH elution buffer may have a pH between about 8.2 and
about 8.8, or between about 8.4 and about 8.6, or a pH of about 8.0, 8.1,
8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0. In a preferred
embodiment, the pH of the elution buffer is about 8.5±0.1.

[0138] In certain embodiments, the eluate from the cation exchange column
may be adjusted to a lower pH, for example between about 5.5 and about
6.5, and diluted with an appropriate buffer such that the conductivity of
the solution is reduced. In certain embodiments, the pH of the cation
exchange eluate may be adjusted to a pH between about 5.7 and about 6.3,
or between about 5.9 and about 6.1, or a pH of about 5.5, 5.6, 5.7, 5.8,
5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. In a preferred embodiment, the pH
of the eluate is adjusted to a pH of about 6.0±0.1. The eluate is then
loaded onto an anion exchange column, which binds several contaminants
found in the preparation. The column flow through, containing the IgG
fraction, is collected during column loading and washing. In certain
embodiments, the ion exchange chromatographic steps of the present
invention can be performed in column mode, batch mode, or in a
combination of the two.

[0139] In certain embodiments, a process improvement is realized by adding
the solution used to adjust the pH by spraying, rather than by fluent
addition.

[0140] 11. Nanofiltration and Ultra/Diafiltration

[0141] In order to further reduce the viral load of the IgG composition
provided herein, the anion exchange column effluent may be nanofiltered
using a suitable nanofiltration device. In certain embodiments, the
nanofiltration device will have a mean pore size of between about 15 nm
and about 200 nm. Examples of nanofilters suitable for this use include,
without limitation, DVD, DV 50, DV 20 (Pall), Viresolve NFP, Viresolve
NFR (Millipore), Planova 15N, 20N, 35N, and 75N (Planova). In a specific
embodiment, the nanofilter may have a mean pore size of between about 15
nm and about 72 nm, or between about 19 nm and about 35 nm, or of about
15 nm, 19 nm, 35 nm, or 72 nm. In a preferred embodiment, the nanofilter
will have a mean pore size of about 35 nm, such as an Asahi PLANOVA 35N
filter or equivalent thereof.

[0142] Optionally, ultrafiltration/diafiltration may performed to further
concentrate the nanofiltrate. In one embodiment, an open channel membrane
is used with a specifically designed post-wash and formulation near the
end the production process render the resulting IgG compositions about
twice as high in protein concentration (200 mg/mL) compared to state of
the art IVIGs (e.g., GAMMAGARD® LIQUID) without affecting yield and
storage stability. With most of the commercial available ultrafiltration
membranes a concentration of 200 mg/mL IgG cannot be reached without
major protein losses. These membranes will be blocked early and therefore
adequate post-wash is difficult to achieve. Therefore open channel
membrane configurations have to be used. Even with open channel
membranes, a specifically designed post-wash procedure has to be used to
obtain the required concentration without significant protein loss (less
than 2% loss). Even more surprising is the fact that the higher protein
concentration of 200 mg/mL does not effect the virus inactivation
capacity of the low pH storage step.

[0143] Subsequent to nanofiltration, the filtrate may be further
concentrated by ultrafiltration/diafiltration. In one embodiment, the
nanofiltrate may be concentrated by ultrafiltration to a protein
concentration of between about 2% and about 10% (w/v). In certain
embodiments, the ultrafiltration is carried out in a cassette with an
open channel screen and the ultrafiltration membrane has a nominal
molecular weight cut off (NMWCO) of less than about 100 kDa or less than
about 90, 80, 70, 60, 50, 40, 30, or fewer kDa. In a preferred
embodiment, the ultrafiltration membrane has a NMWCO of no more than 50
kDa.

[0144] Upon completion of the ultrafiltration step, the concentrate may
further be concentrated via diafiltration against a solution suitable for
intravenous or intramuscular administration. In certain embodiments, the
diafiltration solution may comprise a stabilizing and/or buffering agent.
In a preferred embodiment, the stabilizing and buffering agent is glycine
at an appropriate concentration, for example between about 0.20 M and
about 0.30M, or between about 0.22M and about 0.28M, or between about
0.24M and about 0.26 mM, or at a concentration of about 2.0, 2.1, 2.2,
2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0. In a preferred embodiment, the
diafiltration buffer contains at or about 0.25 M glycine.

[0145] Typically, the minimum exchange volume is at least about 3 times
the original concentrate volume or at least about 4, 5, 6, 7, 8, 9, or
more times the original concentrate volume. The IgG solution may be
concentrated to a final protein concentration of between about 5% and
about 25% (w/v), or between about 6% and about 18% (w/v), or between
about 7% and about 16% (w/v), or between about 8% and about 14% (w/v), or
between about 9% and about 12%, or to a final concentration of about 5%,
or 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,
21%, 22%, 23%, 24%, 25% or higher. In one embodiment, a final protein
concentration of at least about 23% is achieved without adding the
post-wash fraction to the concentrated solution. In another embodiment, a
final protein concentration of at least about 24% is achieved without
adding the post-wash fraction to the concentrated solution. a final
protein concentration of at least about 25% is achieved without adding
the post-wash fraction to the concentrated solution. Typically, at the
end of the concentration process, the pH of the solution will be between
about 4.6 to 5.1.

[0146] In an exemplary embodiment, the pH of the IgG composition is
adjusted to about 4.5 prior to ultrafiltration. The solution is
concentrated to a protein concentration of 5±2% w/v through
ultrafiltration. The UF membrane has a nominal molecular weight cut off
(NMWCO) of 50,000 Daltons or less (Millipore Pellicon Polyether sulfon
membrane). The concentrate is diafiltered against ten volumes of 0.25 M
glycine solution, pH 4.5±0.2. Throughout the ultra-diafiltration
operation the solution is maintained at a temperature of between about
2° C. to about 8° C. After diafiltration, the solution is
concentrated to a protein concentration of at least 11% (w/v).

[0147] 12. Formulation

[0148] Upon completion of the diafiltration step, the protein
concentration of the solution is adjusted to with the diafiltration
buffer to a final concentration of between about 5% and about 20% (w/v),
or between about 6% and about 18% (w/v), or between about 7% and about
16% (w/v), or between about 8% and about 14% (w/v), or between about 9%
and about 12%, or to a final concentration of about 5%, or 6%, 7%, 8%,
9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In a
preferred embodiment, the final protein concentration of the solution is
between about 9% and about 11%, more preferably about 10%.

[0149] The formulated bulk solution is further sterilized by filtering
through a membrane filter with an absolute pore size of no more than
about 0.22 micron, for example about 0.2 micron. Then the solution is
aseptically dispensed into final containers for proper sealing, with
samples taken for testing.

[0150] In one embodiment, the IgG composition is further adjusted to a
concentration of about 10.2±0.2% (w/v) with diafiltration buffer. The
pH is adjusted to about 4.4 to about 4.9 if necessary. Finally, the
solution is sterile filtered and incubated for three weeks at or about
30° C.

[0151] 13. Alcohol Addition

[0152] Advantageously, it has been found that, for purposes of
fractionating IgG from plasma, addition of alcohol by spraying rather
than fluent addition results in reduced loss of IgG yields. Without being
bound by theory, during fluent addition to a plasma fraction, transient
local overconcentration of alcohol at the fluid ingress may lead to
protein denaturation and irreversible loss and/or precipitation of IgG
during steps in which IgG should remain in the supernatant. Furthermore,
these effects may by amplified when large volumes of alcohol need to be
added, such as in industrial scale purifications involving the
fractionation of at least 100 L of pooled plasma.

[0153] The effect of alcohol addition via spraying is exemplified in
Example 14, in which cryo-poor plasma samples are precipitated with 8%
ethanol introduced by either fluent addition (1 and 2) or spray addition
(3 and 4). As can be seen in Table 14, nearly 100% of the IgG present in
the cryo-poor plasma is recovered in the supernatant when ethanol is
added to the sample by spraying, while 4 to 5% of the IgG is lost upon
fluent addition of alcohol. This results in an IgG loss of between about
0.20 and 0.25 g/L at this step alone. In terms of 2007 production levels,
this translates into a loss of about 5.3 million grams (5,300 kilograms)
of IgG. Given the current market price for IVIG, which ranges from
between $50 and $100 per gram, a 4 to 5% loss at this step represents an
global economic loss of up to a half billion dollars annually.

[0154] Accordingly, in one aspect of the methods provided herein, one or
more precipitation steps are performed by the spray addition of alcohol.
In certain embodiments, spray addition may be performed by using any
pressurized device, such as a container (e.g., a spray bottle), that has
a spray head or a nozzle and is operated manually or automatically to
generate a fine mist from a liquid. In certain embodiments, spray
addition is performed while the system is continuously stirred or
otherwise mixed to ensure rapid and equal distribution of the liquid
within the system.

[0155] 14. Adjustment of pH

[0156] The protein precipitation profiles of plasma fractions is highly
dependent upon the pH of the solution from which the plasma proteins are
being precipitated. This fact has been exploited by scientists
fractionating plasma proteins since the introduction of the Cohn and
Oncley methods in 1946 and 1949, respectively. Traditionally, the pH of a
plasma fraction is adjusted prior to alcohol addition to facilitate the
highest recovery yields for the component of interest. Advantageously, it
has now been found that by adjusting the pH of the solution directly
after addition of alcohol or concomitant with alcohol addition results in
a more defined and reproducible precipitation. It was found that ethanol
addition to plasma fractions results in fluctuations in the pH of the
solution, generally by raising the pH of the solution. As such, by
adjusting the pH of a plasma fraction to a predetermined pH before but
not after alcohol addition, the precipitation reaction will occur at a
non-optimal pH.

[0157] Likewise, precipitation of proteins from a plasma fraction will
effect the electrostatic environment and will thus alter the pH of the
solution. Accordingly, as a precipitation event is allowed to progress,
the pH of the solution will begin to diverge from the predetermined pH
value that allows for maximal recovery of the protein species of
interest. This is especially true for precipitation events in which a
large fraction of the protein is being precipitated, precipitation events
in which a high alcohol content is used, and precipitation events that
require a long incubation period.

[0158] The effect of adjusting the pH of a plasma fraction are exemplified
by the results found in Example 16. In this example, IgG was precipitated
from two samples of a Supernatant I fraction after spray addition of
alcohol. The pH of both samples was adjusted to 6.7 before alcohol
addition and readjusted to 6.9 after alcohol addition but prior to the 10
hour precipitation incubation step. In the first sample (reference), the
pH was not adjusted during the 10 hour incubation, while in sample two
(continuous adjustment), the pH was constantly adjusted to pH 6.9 during
the 10 hour incubation. As can be seen in Table 16, after removal of the
modified Fraction II+III precipitate from the samples, the first
supernatant contained 0.2 g IgG/L plasma, while the second sample, in
which the pH was held constant during the precipitation incubation,
contained only 0.13 g IgG/L plasma. The reduced loss of 0.07 g IgG/L
plasma in the second sample represents, in terms of 2007 production
levels, a loss of about 1.9 million grams (1,900 kilograms) of IgG. Given
the current market price for IVIG, which ranges from between $50 and $100
per gram, a 1.5% loss at this step represents an global economic loss of
up to $200 million dollars annually.

[0159] Accordingly, in one aspect of the methods provided herein, the pH
of a plasma fraction is adjusted directly after the addition of alcohol.
In related embodiments, the pH may be adjusted before and after alcohol
addition, or during and after alcohol addition, or before, during, and
after alcohol addition. In a related embodiment, the pH of a solution is
continuously adjusted during one or more alcohol precipitation events or
incubations. In certain embodiments, the pH of a solution is continuously
adjusted or maintained while the system is continuously stirred or
otherwise mixed to ensure rapid and equal distribution of the pH
modifying agent within the system.

[0160] Similar to the case of fluent alcohol addition, it has now been
found that the fluent addition of large volumes of a pH modifying agent
may cause transient local pH variations, resulting in unwanted protein
denaturation or precipitation. Accordingly, in one embodiment of the
methods provided herein, pH modifying agents may be introduced into one
or more plasma fractionation steps by spray addition. In another
embodiment of the methods provided herein, the pH of a plasma fraction or
precipitation step may be adjusted by spray addition of a pH modifying
agent. In certain embodiments, spray addition may be performed by using
any pressurized device, such as a container (e.g., a spray bottle), that
has a spray head or a nozzle and is operated manually or automatically to
generate a fine mist from a liquid. In certain embodiments, spray
addition is performed while the system is continuously stirred or
otherwise mixed to ensure rapid and equal distribution of the liquid
within the system.

III. Concentrated IgG Compositions

[0161] IVIG compositions comprising whole antibodies have been described
for the treatment of certain autoimmune conditions. (See, e.g., U.S.
Patent Publication Nos. US 2002/0114802, US 2003/0099635, and US
2002/0098182.) The IVIG compositions disclosed in these references
include polyclonal antibodies.

[0162] 1. Aqueous IgG Compositions

[0163] In one aspect, the present invention relates to aqueous IgG
compositions prepared by the methods provided herein. Generally, the IgG
compositions prepared by the novel methods described herein will have
high IgG content and purity. For example, IgG compositions provided
herein may have a protein concentration of at least about 3% (w/v) and an
IgG content of greater than about 90% purity. These high purity IgG
compositions are suitable for therapeutic administration, e.g., for IVIG
therapy. In one embodiment, the concentration of IgG is about 10% and is
used for intravenous administration. In another embodiment, the
concentration is about 20% and is used for subcutaneous or intramuscular
administration.

[0164] In one embodiment, the present invention provides an aqueous IgG
composition prepared by a method comprising the steps of (a)
precipitating a cryo-poor plasmid fraction, in a first precipitation
step, with between about 6% and about 10% alcohol at a pH of between
about 6.7 and about 7.3 to obtain a supernatant enriched in IgG, (b)
precipitating IgG from the supernatant with between about 20% and about
30% alcohol at a pH of between about 6.7 and about 7.3 to form a first
precipitate, (c) re-suspending the first precipitate formed in step (b)
to form a suspension, (d) treating the suspension formed in step (c) with
a detergent, (e) precipitating IgG from the suspension with between about
20% and about 30% alcohol at a pH of between about 6.7 and about 7.3 to
form a second precipitate, (f) re-suspending the second precipitate
formed in step (e) to form a suspension, (g) treating the suspension
formed in step (f) with a solvent and/or detergent, and (h) performing at
least one ion exchange chromatography fractionation thereby preparing a
composition of concentrated IgG.

[0165] In a specific embodiment, an IgG composition is provided that is
prepared by a method comprising the steps of (a) adjusting the pH of a
cryo-poor plasma fraction to about 7.0, (b) adjusting the ethanol
concentration of the cryo-poor plasma fraction of step (a) to about 25%
(v/v) at a temperature between about -5° C. and about -9°
C., thereby forming a mixture, (c) separating liquid and precipitate from
the mixture of step (b), (d) re-suspending the precipitate of step (c)
with a buffer containing phosphate and acetate, wherein the pH of the
buffer is adjusted with 600 ml of glacial acetic acid per 1000 L of
buffer, thereby forming a suspension, (e) mixing finely divided silicon
dioxide (SiO2) with the suspension from step (d) for at least about 30
minutes, (f) filtering the suspension with a filter press, thereby
forming a filtrate, (g) washing the filter press with at least 3 filter
press dead volumes of a buffer containing phosphate and acetate, wherein
the pH of the buffer is adjusted with 150 ml of glacial acetic acid per
1000 L of buffer, thereby forming a wash solution, (h) combining the
filtrate of step (f) with the wash solution of step (g), thereby forming
a solution, and treating the solution with a detergent, (i) adjusting the
pH of the solution of step (h) to about 7.0 and adding ethanol to a final
concentration of about 25%, thereby forming a precipitate, (j) separating
liquid and precipitate from the mixture of step (i), (k) dissolving the
precipitate in an aqueous solution comprising a solvent or detergent and
maintaining the solution for at least 60 minutes, (l) passing the
solution after step (k) through a cation exchange chromatography column
and eluting proteins absorbed on the column in an eluate, (m) passing the
eluate from step (l) through an anion exchange chromatography column to
generate an effluent, (n) passing the effluent from step (m) through a
nanofilter to generate a nanofiltrate, (o) passing the nanofiltrate from
step (n) through an ultrafiltration membrane to generate an
ultrafiltrate, and (p) diafiltrating the ultrafiltrate from step (o)
against a diafiltration buffer to generate a diafiltrate having a protein
concentration between about 8% (w/v) and about 12% (w/v), thereby
obtaining a composition of concentrated IgG.

[0166] In certain embodiments, aqueous IgG compositions are prepared using
a method provided herein that comprises improvements in two or more of
the fractionation process steps described above. For example, in certain
embodiments the improvements may be found in the first precipitation
step, the Modified Fraction II+III precipitation step, the Modified
Fraction II+III dissolution step, and/or the Modified Fraction II+III
suspension filtration step.

[0167] In one embodiment, an aqueous IgG composition is provided that is
prepared by a purification method described herein, wherein the method
comprises the spray addition of one or more solutions that would
otherwise be introduced into a plasma fraction by fluent addition. For
example, in certain embodiments the method will comprise the introduction
of alcohol (e.g., ethanol) into a plasma fraction by spraying. In other
embodiments, solutions that may be added to a plasma fraction by spraying
include, without limitation, a pH modifying solution, a solvent solution,
a detergent solution, a dilution buffer, a conductivity modifying
solution, and the like. In a preferred embodiment, one or more alcohol
precipitation steps is performed by the addition of alcohol to a plasma
fraction by spraying. In a second preferred embodiment, one or more pH
adjustment steps is performed by the addition of a pH modifying solution
to a plasma fraction by spraying.

[0168] In certain embodiments, an aqueous IgG composition is provided that
is prepared by a purification method described herein, wherein the method
comprises adjusting the pH of a plasma fraction being precipitated after
and/or concomitant with the addition of the precipitating agent (e.g.,
alcohol or polyethelene glycol). In some embodiments, a process
improvement is provided in which the pH of a plasma fraction being
actively precipitated is maintained throughout the entire precipitation
incubation or hold step by continuous monitoring and adjustment of the
pH. In preferred embodiments the adjustment of the pH is performed by the
spray addition of a pH modifying solution.

[0169] In one embodiment, the present invention provides aqueous IgG
compositions comprising a protein concentration of between about 30 g/L
and about 250 g/L. In certain embodiments, the protein concentration of
the IgG composition is between about 50 g/L and about 200 g/L, or between
about 70 g/L and about 150 g/L, or between about 90 g/L and about 120
g/L, or any suitable concentration within these ranges, for example about
30 g/L, or about 35 g/L, 40 g/L, 45 g/L, 50 g/L, 55 g/L, 60 g/L, 65 g/L,
70 g/L, 75 g/L, 80 g/L, 85 g/L, 90 g/L, 95 g/L, 100 g/L, 105 g/L, 110
g/L, 115 g/L, 120 g/L, 125 g/L, 130 g/L, 135 g/L, 140 g/L, 145 g/L, 150
g/L, 155 g/L, 160 g/L, 165 g/L, 170 g/L, 175 g/L, 180 g/L, 185 g/L, 190
g/L, 195 g/L, 200 g/L, 205 g/L, 210 g/L, 215 g/L, 220 g/L, 225 g/L, 230
g/L, 235 g/L, 240 g/L, 245 g/L, 250 g/L, or higher. In a preferred
embodiment, the aqueous IgG composition will have a concentration of at
or about 10%. In a particularly preferred embodiment, the composition
will have a concentration of 10.2±0.2% (w/v) In another preferred
embodiment, the aqueous IgG composition will have a concentration of at
or about 20%.

[0170] The methods provided herein allow for the preparation of IgG
compositions having very high levels of purity. In one embodiment, at
least about 95% of the total protein in a composition provided herein
will be IgG. In other embodiments, at least about 96% of the protein is
IgG, or at least about 97%, 98%, 99%, 99.5%, or more of the total protein
of the composition will be IgG. In a preferred embodiment, at least 97%
of the total protein of the composition will be IgG. In another preferred
embodiment, at least 98% of the total protein of the composition will be
IgG. In another preferred embodiment, at least 99% of the total protein
of the composition will be IgG.

[0171] Similarly, the methods provided herein allow for the preparation of
IgG compositions which containing extremely low levels of contaminating
agents. For example, Table 19 provides the results of impurity testing
for three bulk solutions of IgG prepared by the improved methods provided
herein. In certain embodiments, IgG compositions are provided that
contain less than about 140 mg/L IgA. In other embodiments, the IgG
composition will contain less than about 60 mg/L IgA, preferably less
than about 40 mg/L IgA, most preferably less than about 30 mg/L IgA.

[0172] In another embodiment, IgG compositions are provided that contain
less than about 50 mg/L IgM. In other embodiments, the IgG composition
will contain less than about 25 mg/L IgM, preferably less than about 10
mg/L IgM, more preferably less than about 5 mg/L IgM, more preferably
less than about 4 mg/L IgM, more preferably less than about 3 mg/L IgM,
most preferably less than about 2.5 mg/L IgM.

[0173] In another embodiment, IgG compositions are provided that contain
less than about 100 PL-1 nmol/mL min amidolytic activity. In other
embodiments, the IgG composition will contain less than about 50 PL-1
nmol/mL min amidolytic activity, preferably less than about 25 PL-1
nmol/mL min amidolytic activity, more preferably less than about 20 PL-1
nmol/mL min amidolytic activity, more preferably less than about 15 PL-1
nmol/mL min amidolytic activity, most preferably less than about 10 PL-1
nmol/mL min amidolytic activity.

[0174] In another embodiment, IgG compositions are provided that contain
less than about 20 mg/L fibrinogen. In other embodiments, the IgG
composition will contain less than about 10 mg/L fibrinogen, preferably
less than about 5 mg/L fibrinogen, more preferably less than about 2.5
mg/L fibrinogen, more preferably less than about 1 mg/L fibrinogen, more
preferably less than about 0.5 mg/L fibrinogen, most preferably less than
about 0.25 mg/L fibrinogen.

[0175] In yet another embodiment, IgG compositions are provided that
consist of primarily IgG monomers/dimmers. In one embodiment, an IgG
composition is provided in which at least 95%, 96%, 97%, 98%, 99%, 99.1%,
99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% of the IgG is
monomeric or dimeric. In a preferred embodiment, an IgG composition is
provided in which at least 97% of the IgG is monomeric or dimeric. In a
more preferred embodiment, at least 99% of the IgG is monomeric or
dimeric. In a more preferred embodiment, at least 99.5% of the IgG is
monomeric or dimeric. In a more preferred embodiment, at least 99.7% of
the IgG is monomeric or dimeric.

[0176] 2. Pharmaceutical Compositions

[0177] In another aspect, the present invention provides pharmaceutical
compositions and formulations comprising purified IgG prepared by the
methods provided herein. Generally, the IgG pharmaceutical compositions
and formulations prepared by the novel methods described herein will have
high IgG content and purity. For example, IgG pharmaceutical compositions
and formulations provided herein may have a protein concentration of at
least about 7% (w/v) and an IgG content of greater than about 95% purity.
These high purity IgG pharmaceutical compositions and formulations are
suitable for therapeutic administration, e.g., for IVIG therapy. In a
preferred embodiment, a pharmaceutical IgG composition is formulated for
intravenous administration (e.g., IVIG therapy).

[0178] In one embodiment, the pharmaceutical compositions provided herein
are prepared by formulating an aqueous IgG composition isolated using a
method provided herein. Generally, the formulated composition will have
been subjected to at least one, preferably at least two, most preferably
at least three, viral inactivation or removal steps. Non-limiting
examples of viral inactivation or removal steps that may be employed with
the methods provided herein include, solvent detergent treatment
(Horowitz et al., Blood Coagul Fibrinolysis 1994 (5 Suppl 3):S21-S28 and
Kreil et al., Transfusion 2003 (43):1023-1028, both of which are herein
expressly incorporated by reference in their entirety for all purposes),
nanofiltration (Hamamoto et al., Vox Sang 1989 (56)230-236 and Yuasa et
al., J Gen Virol. 1991 (72 (pt 8)):2021-2024, both of which are herein
expressly incorporated by reference in their entirety for all purposes),
and low pH incubation at high temperatures (Kempf et al., Transfusion
1991 (31)423-427 and Louie et al., Biologicals 1994 (22):13-19).

[0179] In certain embodiments, pharmaceutical formulations are provided
having an IgG content of between about 80 g/L IgG and about 120 g/L IgG.
Generally, these IVIG formulations are prepared by isolating an IgG
composition from plasma using a method described herein, concentrating
the composition, and formulating the concentrated composition in a
solution suitable for intravenous administration. The IgG compositions
may be concentrated using any suitable method known to one of skill in
the art. In one embodiment, the composition is concentrated by
ultrafiltration/diafiltration. In some embodiments, the ultrafiltration
device used to concentrate the composition will employ an ultrafiltration
membrane having a nominal molecular weight cut off (NMWCO) of less than
about 100 kDa or less than about 90, 80, 70, 60, 50, 40, 30, or fewer
kDa. In a preferred embodiment, the ultrafiltration membrane has a NMWCO
of no more than 50 kDa. Buffer exchange may be achieved using any
suitable technique known to one of skill in the art. In a specific
embodiment, buffer exchange is achieved by diafiltration.

[0180] In one specific embodiment, a pharmaceutical composition of IgG is
provided, wherein the IgG composition was purified from plasma using a
method comprising the steps of (a) precipitating a cryo-poor plasmid
fraction, in a first precipitation step, with between about 6% and about
10% alcohol at a pH of between about 6.7 and about 7.3 to obtain a
supernatant enriched in IgG, (b) precipitating IgG from the supernatant
with between about 20% and about 30% alcohol at a pH of between about 6.7
and about 7.3 to form a first precipitate, (c) re-suspending the first
precipitate formed in step (b) to form a suspension, (d) treating the
suspension formed in step (c) with a detergent, (e) precipitating IgG
from the suspension with between about 20% and about 30% alcohol at a pH
of between about 6.7 and about 7.3 to form a second precipitate, (f)
re-suspending the second precipitate formed in step (e) to form a
suspension, (g) treating the suspension formed in step (f) with a solvent
and/or detergent, (h) performing at least one ion exchange chromatography
fractionation; (i) performing a solvent detergent treatment; and (j)
subjecting the composition to nanofiltration, thereby preparing a
composition of IgG.

[0181] In a specific embodiment, a pharmaceutical composition of IgG is
provided, wherein the IgG composition was purified from plasma using a
method comprising the steps of (a) adjusting the pH of a cryo-poor plasma
fraction to about 7.0, (b) adjusting the ethanol concentration of the
cryo-poor plasma fraction of step (a) to about 25% (v/v) at a temperature
between about -5° C. and about -9° C., thereby forming a
mixture, (c) separating liquid and precipitate from the mixture of step
(b), (d) re-suspending the precipitate of step (c) with a buffer
containing phosphate and acetate, wherein the pH of the buffer is
adjusted with 600 ml of glacial acetic acid per 1000 L of buffer, thereby
forming a suspension, (e) mixing finely divided silicon dioxide (SiO2)
with the suspension from step (d) for at least about 30 minutes, (f)
filtering the suspension with a filter press, thereby forming a filtrate,
(g) washing the filter press with at least 3 filter press dead volumes of
a buffer containing phosphate and acetate, wherein the pH of the buffer
is adjusted with 150 ml of glacial acetic acid per 1000 L of buffer,
thereby forming a wash solution, (h) combining the filtrate of step (f)
with the wash solution of step (g), thereby forming a solution, and
treating the solution with a detergent, (i) adjusting the pH of the
solution of step (h) to about 7.0 and adding ethanol to a final
concentration of about 25%, thereby forming a precipitate, (j) separating
liquid and precipitate from the mixture of step (i), (k) dissolving the
precipitate in an aqueous solution comprising a solvent or detergent and
maintaining the solution for at least 60 minutes, (l) passing the
solution after step (k) through a cation exchange chromatography column
and eluting proteins absorbed on the column in an eluate, (m) passing the
eluate from step (l) through an anion exchange chromatography column to
generate an effluent, (n) passing the effluent from step (m) through a
nanofilter to generate a nanofiltrate, (o) passing the nanofiltrate from
step (n) through an ultrafiltration membrane to generate an
ultrafiltrate, and (p) diafiltrating the ultrafiltrate from step (o)
against a diafiltration buffer to generate a diafiltrate having a protein
concentration between about 8% (w/v) and about 12% (w/v), thereby
obtaining a composition of concentrated IgG.

[0182] In certain embodiments, a pharmaceutical composition of IgG is
provided, wherein the IgG composition is prepared using a method provided
herein that comprises improvements in two or more of the fractionation
process steps described above. For example, in certain embodiments the
improvements may be found in the first precipitation step, the Modified
Fraction II+III precipitation step, the Modified Fraction II+III
dissolution step, and/or the Modified Fraction II+III suspension
filtration step.

[0183] In certain embodiments, a pharmaceutical composition of IgG is
provided, wherein the IgG composition is prepared using a purification
method described herein, wherein the method comprises the spray addition
of one or more solutions that would otherwise be introduced into a plasma
fraction by fluent addition. For example, in certain embodiments the
method will comprise the introduction of alcohol (e.g., ethanol) into a
plasma fraction by spraying. In other embodiments, solutions that may be
added to a plasma fraction by spraying include, without limitation, a pH
modifying solution, a solvent solution, a detergent solution, a dilution
buffer, a conductivity modifying solution, and the like. In a preferred
embodiment, one or more alcohol precipitation steps is performed by the
addition of alcohol to a plasma fraction by spraying. In a second
preferred embodiment, one or more pH adjustment steps is performed by the
addition of a pH modifying solution to a plasma fraction by spraying.

[0184] In certain embodiments, a pharmaceutical composition of IgG is
provided, wherein the IgG composition is prepared by a purification
method described herein, wherein the method comprises adjusting the pH of
a plasma fraction being precipitated after and/or concomitant with the
addition of the precipitating agent (e.g., alcohol or polyethelene
glycol). In some embodiments, a process improvement is provided in which
the pH of a plasma fraction being actively precipitated is maintained
throughout the entire precipitation incubation or hold step by continuous
monitoring and adjustment of the pH. In preferred embodiments the
adjustment of the pH is performed by the spray addition of a pH modifying
solution.

[0185] In one embodiment, the present invention provides a pharmaceutical
composition of IgG comprising a protein concentration of between about 70
g/L and about 130 g/L. In certain embodiments, the protein concentration
of the IgG composition is between about 80 g/L and about 120 g/L,
preferably between about 90 g/L and about 110 g/L, most preferably of
about 100 g/L, or any suitable concentration within these ranges, for
example about 70 g/L, 75 g/L, 80 g/L, 85 g/L, 90 g/L, 95 g/L, 100 g/L,
105 g/L, 110 g/L, 115 g/L, 120 g/L, 125 g/L, or 130 g/L. In a preferred
embodiment, a pharmaceutical composition is provided having a protein
concentration of at or about 100 g/L. In a particularly preferred
embodiment, the pharmaceutical composition will have a protein
concentration of at or about 102 g/L.

[0186] In another embodiment, the present invention provides a
pharmaceutical composition of IgG comprising a protein concentration of
between about 170 g/L and about 230 g/L. In certain embodiments, the
protein concentration of the IgG composition is between about 180 g/L and
about 220 g/L, preferably between about 190 g/L and about 210 g/L, most
preferably of about 200 g/L, or any suitable concentration within these
ranges, for example about 170 g/L, 175 g/L, 180 g/L, 185 g/L, 190 g/L,
195 g/L, 200 g/L, 205 g/L, 210 g/L, 215 g/L, 220 g/L, 225 g/L, or 230
g/L. In a preferred embodiment, a pharmaceutical composition is provided
having a protein concentration of at or about 200 g/L.

[0187] The methods provided herein allow for the preparation of IgG
pharmaceutical compositions having very high levels of purity. For
example, in one embodiment, at least about 95% of the total protein in a
composition provided herein will be IgG. In other embodiments, at least
about 96% of the protein is IgG, or at least about 97%, 98%, 99%, 99.5%,
or more of the total protein of the composition will be IgG. In a
preferred embodiment, at least 97% of the total protein of the
composition will be IgG. In another preferred embodiment, at least 98% of
the total protein of the composition will be IgG. In another preferred
embodiment, at least 99% of the total protein of the composition will be
IgG.

[0188] Similarly, the methods provided herein allow for the preparation of
IgG pharmaceutical compositions which containing extremely low levels of
contaminating agents. For example, in certain embodiments, IgG
compositions are provided that contain less than about 100 mg/L IgA. In
other embodiments, the IgG composition will contain less than about 50
mg/L IgA, preferably less than about 35 mg/L IgA, most preferably less
than about 20 mg/L IgA.

[0189] The pharmaceutical compositions provided herein will typically
comprise one or more buffering agents or pH stabilizing agents suitable
for intravenous, subcutaneous, and/or intramuscular administration.
Non-limiting examples of buffering agents suitable for formulating an IgG
composition provided herein include glycine, citrate, phosphate, acetate,
glutamate, tartrate, benzoate, lactate, histidine or other amino acids,
gluconate, malate, succinate, formate, propionate, carbonate, or any
combination thereof adjusted to an appropriate pH. Generally, the
buffering agent will be sufficient to maintain a suitable pH in the
formulation for an extended period of time. In a preferred embodiment,
the buffering agent is glycine.

[0190] In some embodiments, the concentration of buffering agent in the
formulation will be between about 100 mM and about 400 mM, preferably
between about 150 mM and about 350 mM, more preferably between about 200
mM and about 300 mM, most preferably about 250 mM. In a particularly
preferred embodiment, the IVIG composition will comprise between about
200 mM and about 300 mM glycine, most preferably about 250 mM glycine.

[0191] In certain embodiments, the pH of the formulation will be between
about 4.1 and about 5.6, preferably between about 4.4 and about 5.3, most
preferably between about 4.6 and about 5.1. In particular embodiments,
the pH of the formulation may be about 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,
4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, or 5.6. In a preferred
embodiment, the pH of the formulation will be between about 4.6 and about
5.1.

[0194] The IgG formulations provided herein are generally stable in liquid
form for an extended period of time. In certain embodiments, the
formulations are stable for at least about 3 months at room temperature,
or at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, or 24 months at room temperature. The formulation
will also generally be stable 6or at least about 18 months under
refrigerated conditions (typically between about 2° C. and about
8° C.), or for at least about 21, 24, 27, 30, 33, 36, 39, 42, or
45 months under refrigerated conditions.

IV. Methods of Treatment

[0195] As routinely practiced in the modern medicine, sterilized
preparations of concentrated immunoglobulins (especially IgGs) are used
for treating medical conditions that fall into these three main classes:
immune deficiencies, inflammatory and autoimmune diseases, and acute
infections. These IgG preparations may also be useful for treating
multiple sclerosis (especially relapsing-remitting multiple sclerosis or
RRMS), Alzheimer's disease, and Parkinson's disease. The purified IgG
preparation of this invention is suitable for these purposes, as well as
other clinically accepted uses of IgG preparations.

[0196] The FDA has approved the use of IVIG to treat various indications,
including allogeneic bone marrow transplant, chronic lymphocytic
leukemia, idiopathic thrombocytopenic purpura (ITP), pediatric HIV,
primary immunodeficiencies, Kawasaki disease, chronic inflammatory
demyelinating polyneuropathy (CIDP), and kidney transplant with a high
antibody recipient or with an ABO incompatible donor. In certain
embodiments, the IVIG compositions provided herein are useful for the
treatment or management of these diseases and conditions.

[0198] Finally, experimental use of IVIG for the treatment or management
of diseases including primary immune deficiency, RRMS, Alzheimer's
disease, and Parkinson's disease has been proposed (U.S. Patent
Application Publication No. U.S. 2009/0148463, which is herein
incorporated by reference in its entirety for all purposes). In certain
embodiments, the IVIG compositions provided herein are useful for the
treatment or management of primary immune deficiency, RRMS, Alzheimer's
disease, or Parkinson's disease. In certain embodiments comprising daily
administration, an effective amount to be administered to the subject can
be determined by a physician with consideration of individual differences
in age, weight, disease severity, route of administration (e.g.,
intravenous v. subcutaneous) and response to the therapy. In certain
embodiments, an immunoglobulin preparation of this invention can be
administered to a subject at about 5 mg/kilogram to about 2000
mg/kilogram each day. In additional embodiments, the immunoglobulin
preparation can be administered in amounts of at least about 10
mg/kilogram, at last 15 mg/kilogram, at least 20 mg/kilogram, at least 25
mg/kilogram, at least 30 mg/kilogram, or at least 50 mg/kilogram. In
additional embodiments, the immunoglobulin preparation can be
administered to a subject at doses up to about 100 mg/kilogram, to about
150 mg/kilogram, to about 200 mg/kilogram, to about 250 mg/kilogram, to
about 300 mg/kilogram, to about 400 mg/kilogram each day. In other
embodiments, the doses of the immunoglobulin preparation can be greater
or less. Further, the immunoglobulin preparations can be administered in
one or more doses per day. Clinicians familiar with the diseases treated
by IgG preparations can determine the appropriate dose for a patient
according to criteria known in the art.

[0199] In accordance with the present invention, the time needed to
complete a course of the treatment can be determined by a physician and
may range from as short as one day to more than a month. In certain
embodiments, a course of treatment can be from 1 to 6 months.

[0200] An effective amount of an IVIG preparation is administered to the
subject by intravenous means. The term "effective amount" refers to an
amount of an IVIG preparation that results in an improvement or
remediation of disease or condition in the subject. An effective amount
to be administered to the subject can be determined by a physician with
consideration of individual differences in age, weight, the disease or
condition being treated, disease severity and response to the therapy. In
certain embodiments, an IVIG preparation can be administered to a subject
at dose of about 5 mg/kilogram to about 2000 mg/kilogram per
administration. In certain embodiments, the dose may be at least about 5
mg/kg, or at least about 10 mg/kg, or at least about 20 mg/kg, 30 mg/kg,
40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg,
125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350
mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 650 mg/kg,
700 mg/kg, 750 mg/kg, 800 mg/kg, 850 mg/kg, 900 mg/kg, 950 mg/kg, 1000
mg/kg, 1100 mg/kg, 1200 mg/kg, 1300 mg/kg, 1400 mg/kg, 1500 mg/kg, 1600
mg/kg, 1700 mg/kg, 1800 mg/kg, 1900 mg/kg, or at least about 2000 mg/kg.

[0201] The dosage and frequency of IVIG treatment will depend upon, among
other factors. the disease or condition being treated and the severity of
the disease or condition in the patient. Generally, for primary immune
dysfunction a dose of between about 100 mg/kg and about 400 mg/kg body
weight will be administered about every 3 to 4 weeks. For neurological
and autoimmune diseases, up to 2 g/kg body weight is implemented for
three to six months over a five day course once a month. This is
generally supplemented with maintenance therapy comprising the
administration of between about 100 mg/kg and about 400 mg/kg body weight
about once every 3 to 4 weeks. Generally, a patient will receive a dose
or treatment about once every 14 to 35 days, or about every 21 to 28
days. The frequency of treatment will depend upon, among other factors.
the disease or condition being treated and the severity of the disease or
condition in the patient.

[0202] In a preferred embodiment, a method of treating an
immunodeficiency, autoimmune disease, or acute infection in a human in
need thereof is provided, the method comprising administering a
pharmaceutical IVIG composition of the present invention. In a related
embodiment, the present invention provides IVIG compositions manufactured
according to a method provided herein for the treatment of an
immunodeficiency, autoimmune disease, or acute infection in a human in
need thereof.

[0204] The following examples are provided by way of illustration only and
not by way of limitation. Those of skill in the art will readily
recognize a variety of non-critical parameters that could be changed or
modified to yield essentially the same or similar results.

Example 1

[0205] The present example demonstrates that significant amounts of
fibrinogen, amidolytic activity, prekallikrein activity, and lipoproteins
can be removed from an extracted modified Fraction II+III paste
suspension by treatment with Aerosil prior to filtration.

[0206] Fumed silica (Aerosil 380) is currently used to adsorb fibrinogen,
amidolytic activity, prekallikrein activity, and lipoproteins. To
investigate the effect of Aerosil in more detail, six modified Fraction
II+III suspensions were treated with varying amounts of Aerosil prior to
filtration Briefly, dissolution buffer containing 5 mM sodium acetate/5
mM sodium dihydrogen phosphate buffer pH 4.5 was used to re-suspend
modified II+III paste, prepared as described herein, at a ratio of 15
grams dissolution buffer per gram II+III paste. After paste addition, the
suspension was stirred for one hour at between 2° C. and 8°
C. in a pH controlled environment (pH IPC limits: 4.9 to 5.3). We have
found that the pH of this suspension normally shifts to a pH of about
5.1, and thus further pH adjustment is not necessary. After an additional
extraction, for at least 120 minutes, Aerosil 380, at between 0 and 100
mg per gram II+III paste, was added to the containers and the suspensions
were incubated for one hour. Diatomaceous earth was added prior to depth
filtration with a Cuno 50SA filter. After filtration, the filters were
washed with extraction buffer containing 8 g L-1 citrate and 0.2%
polysorbate 80 (pH 5.0) and the wash was added to the filtrate. The
combined filtrate and wash solution was then treated with Polysorbate 80
to further solubilize hydrophobic impurities, for example lipoproteins,
and the IgG was precipitated with 25% ethanol (pH 7) at between
-8° C. and -10° C. The resulting Ppt G precipitate was
almost white and possessed higher IgG purity. The precipitate was then
dissolved in purified water at a ratio of 7 grams water per 2 grams Ppt G
precipitate.

[0207] The IgG solutions were analyzed for IgG recovery and impurities
following the Cuno filtration step. Specifically, levels of amidolytic
activity (PL-1), PKA activity, and fibrinogen were measured (Table 3).
Notably, as seen in Table 3, extraction protocols using 40 to 60 mg
Aerosil 380 per g II+III paste resulted in acceptable levels of IgG
recovery with significant decreases in amidolytic and PKA activity, as
well as a significant decrease in the level of fibrinogen in the
filtrate. Compared to extractions performed without Aerosil treatment,
the addition of 40 mg Aerosil 380 per gram II+III paste resulted in an
almost 90% reduction of PKA activity and fibrinogen content and a 60%
reduction of amidolytic activity, while maintaining similar IgG recovery
(73%).

[0208] The present example demonstrates that significant amounts of
fibrinogen can be removed from an extracted modified Fraction II+III
paste suspension by treatment with Aerosil prior to filtration. One
purpose of the present experiment was to find suitable conditions for
efficient fibrinogen removal without incurring significant losses of IgG.

[0209] Modified II+III paste, prepared according to the method provided
herein, was dissolved in 5 mM sodium acetate/5 mM monobasic sodium
phosphate buffer pH 4.5. The dissolution ratio was 15 kg of buffer per 1
kg II+III paste. The amount of acetic acid added to the buffer was chosen
in a way that pH after sixty minutes stirring was 4.9. In order to fully
homogenize the suspension, it was stirred up to twenty hours at 2 to
8° C. before being separated into 6 portions of 50 ml each in 100
ml beakers, where varying amounts of Aerosil 380 were already present, as
given in Table 2. The II+III suspension solutions were then stirred for
80 minutes in the presence of the Aerosil, prior to processing and
analysis. After stirring all samples were centrifuged with a Heraeus
Cryofuge 8500i at 4600 RPM for 30 minutes at 4° C. in 50 ml falcon
tubes.

[0210] In this experiment, IgG measurements were taken using the
nephelometric test, which was chosen due to the more accurate values,
compared to the ELISA test, at the high concentrations found in II+III
suspension solutions. To minimize the irritation of unspecific turbidity,
the samples were filtered through 0.45 μm filters prior to testing.
For IgM, IgA, and fibrinogen, ELISA tests were preferred due to the lower
concentrations of these impurities in the suspension. The results of the
experiment are shown below in Table 2.

[0211] To further characterize the effect of Aerosil treatment on
fibrinogen removal and IgG loss as described in Example 1, Aerosil
concentrations were further titrated between 0 mg and 40 mg per gram
modified II+III paste. The results shown in Table 2 confirm the high
capacity of Aerosil to reduce fibrinogen in this fraction. Notably, use
of 40 mg per gram II+III paste results in almost 90% reduction of
fibrinogen, while only reducing IgG recovery in the filter cake by 10%.

[0212] The present example demonstrates suitable conditions that allow for
highly efficient extraction of IgG from a modified Fraction II+III paste,
while limiting the levels of detrimental impurities. Specifically,
parameters including the concentration of acetic acid used in the II+III
dissolution buffer and aerosil treatment of the extracted solution prior
to filtration were examined.

[0213] II+III paste was extracted in 5 mM sodium acetate, 5 mM sodium
dihydrogen phosphate and variable amounts of concentrated acetic acid, as
shown in Table 1, for 180 minutes at between 2° C. and 8°
C., followed by addition of Aerosil 380 as shown in Table 1. After one
hour of stirring, the suspension was clarified by Cuno 50SA filtration in
the presence of diatomaceous earth. Post-wash of the filter was carried
out with the same buffer as for the extraction except the different
amount of acetic acid, as given in Table 1, using 40% of the volume of
the suspension prior to filtration. Precipitate G precipitation in the
presence of 25% ethyl alcohol; 8 g L-1 sodium citrate, and 0.2%
Tween 80 (pH 7) at -8° C. was performed and after 8 hours hold
time, separation was performed by centrifugation with Heraeus Cryofuge
8500i in stainless steel beakers at 4600 RPM for 30 minutes at
-10° C. The precipitate was dissolved in a 1:2 ratio in purified
water.

[0214] As can be seen in Table 1, Aerosil addition to the II+III paste
suspension has a marked influence on γ-globulin purity in the Ppt G
fraction. Without Aerosil the γ-globulin purity is only 83.5%,
while addition of 40 mg Aerosil per gram II+III paste to the II+III paste
suspension increases the γ-globulin purity to 87.2%. As evidenced,
Aerosil treatment leads to a significant reduction of fibrinogen, PKA and
amidolytic activity. One drawback of the impurity adsorption on Aerosil
is that IgG loss in the filter cake is increased with increasing amounts
of Aerosil. However, as shown in Table 1, higher concentrations of acetic
acid in the extraction buffer partially counterbalances the effect of
Aerosil on IgG loss in the filter cake, and further reduced IgG loss in
the supernatant Ppt G fraction. As can be seen in Table 1, increasing the
amount of acetic acid in the dissolution buffer from 400 μL per L
paste to 510 μL per L paste, reduced the amount of IgG lost in the
filter cake by almost 50%. Advantageously, the higher acetic acid
concentration does not affect the γ-globulin purity in the Ppt G
fraction (87.2% pure using 400 μL per L paste vs. 86.7% pure using 510
μL per L paste). Furthermore, the results show that the difference in
pH value caused by the different amount of acetic acid is negligible, due
to the high buffer capacity of acetic acid near its pka value of 4.75
(Merck). This suggests that for better accuracy in large-scale
manufacturing, acetic acid should be added by weight. Thus, the influence
of Aerosil on purity is much higher than the influence of acetic acid on
purity, in the investigated range, as shown with the γ-globulin
content measured by CAE.

Example 4

[0215] The results found in Example 3 suggested that the amount of IgG
lost in the filter cake is strongly dependent on the amount of acetic
acid used for pH adjustment of the extraction buffer at a given Aerosil
concentration. In order to further characterize this effect, modified
II+III paste was extracted in purified water for about 120 minutes to
obtain a homogeneous suspension and divided into 4 parts. These parts
were adjusted to pH 3.8, 4.2, 4.6, and 5.0, respectively, with 1M acetic
acid followed by a second extraction time for another 120 minutes.
Afterwards, Aerosil treatment was done with 40 mg Aerosil 380 per gram
II+III paste. After one hour stirring the suspension was clarified by
Cuno 50SA filtration in the presence of diatomaceous earth. Post-wash of
the filter was carried out with 100 percent of the volume of the
suspension prior filtration with extraction buffer adjusted to the pH as
given above. The filtrate was treated with 8 g L-1 sodiumcitrate and
0.2% Tween 80, adjusted to pH 7.0, and IgG was precipitated with 25%
alcohol at -8° C. PptG precipitate was recovered by centrifugation
at 4600 RPM for 30 minutes at -10° C. in a Heraeus Cryofuge 8500i
using stainless steel beakers. The precipitate was then dissolved in
purified water at a ratio of 7 grams water per 2 grams Ppt G precipitate.
The relevant fractions were then assayed for IgG recovery, PKA activity,
fibrinogen content, and amidolytic activity, to determine the pH
dependence of recovery (Table 4).

[0216] Table 4 shows that PKA, amidolytic activity (PL-1), and fibrinogen
removal with Aerosil is less effective at lower pH during clarification.
It can be seen from the results obtained here, that the most effective pH
for effective removal of PKA, amidolytic activity (PL-1), and fibrinogen,
while maintaining efficient IgG recovery in the Ppt G fraction, is about
pH 5. High concentrations of acetic acid lead to a significant IgG loss
in Ppt G supernatant (0.85 g L-1 in the presence of 75 mM acetic
acid) while IgG loss in the filter cake is minimized.

Example 5

[0217] To determine the dependence of Aerosil treatment on the results
found in Example 4, the experiment was repeated, but with the Aerosil
treatment step omitted. Briefly, II+III paste was extracted in purified
water for about 120 minutes to obtain a homogeneous suspension and
divided into 4 parts. These parts were adjusted to pH 3.8, 4.2, 4.6, and
5.0, respectively, with 1M acetic acid followed by a second extraction
time for another 120 minutes. Afterwards, the suspension was clarified by
Cuno 50SA filtration in the presence of diatomaceous earth. Post-wash of
the filter was carried out with 100 percent of the volume of the
suspension prior filtration with extraction buffer adjusted to the pH as
given above. The filtrate was treated with 8 g L-1 sodium citrate
and 0.2% Tween 80, adjusted to pH 7.0, and IgG was precipitated with 25%
alcohol at -8° C. Ppt G precipitate was recovered by
centrifugation at 4600 RPM for 30 minutes at -10° C. in a Heraeus
Cryofuge 8500i using stainless steel beakers. The precipitate was then
dissolved in purified water at a ratio of 7 grams water per 2 grams Ppt G
precipitate. The relevant fractions were then assayed for IgG recovery,
PKA activity, fibrinogen content, and amidolytic activity, to determine
the pH dependence of recovery (Table 5).

[0218] Consistent with the results found in Example 4, increasing the pH
of the extraction/dissolution buffer to 5.0 resulted in a small increase
in the IgG lost in the filter cake, however, this loss was more than
offset by a larger decrease in the loss of IgG in the Ppt G supernatant.

[0219] When the results of Example 4 and Example 5 are compared, it can be
seen that Aerosil treatment reduces the amount of residual PKA activity
found in the dissolved PptG fraction when the II+III paste is extracted
at lower pHs (3.8, 4.2, and 4.6), but not at pH 5.0 (FIG. 2). Conversely,
Aerosil treatment significantly reduces the fibrinogen content of the
dissolved Ppt G fraction when the II+III paste is extracted at higher pHs
(FIG. 3; compare pH 4.6 and 5.0 to pH 3.8 and 4.2). Aerosil treatment
does not appear to affect the level of residual amidolytic activity found
in the dissolved Ppt G fraction (FIG. 4). Notably, the level of all three
contaminants in the dissolved Ppt G fraction is considerably reduced when
the II+III paste is extracted at pH 5.0, as compared to pH 3.8, 4.2, and
4.6.

Example 6

[0220] As can be seen in the Examples above, IgG losses in the filter cake
and Ppt G supernatants are minimized when the II+III paste is extracted
at a pH of around 4.5 to 4.6. However, it is also evidenced in the
previous examples, that critical impurities, including PKA activity,
amidolytic activity, and fibrinogen content, are much higher when the
II+III paste is extracted at pH 4.5 or 4.6 compared to when the
extraction occurs at a pH around 4.9 to 5.0. Accordingly, the present
example was performed to determine if the higher impurity levels seen
when the II+III paste is extracted at pH 4.5 could be offset by
increasing the amount of Aerosil used to adsorb contaminants, while
maintaining low levels of IgG loss in the filter cake and Ppt G
supernatant.

[0221] Along these lines, extraction of modified II+III paste was
performed as before, with extraction buffer having a pH of 4.5.
Increasing amounts of Aerosil 380, up to 200 mg per g II+III paste, were
then added and the suspension was stirred for one hour. Further
processing of the sample was performed as above.

[0222] As can be seen in Table 6, both fibrinogen and PKA activity removal
is significantly improved by clarification with high amounts of Aerosil.
IgG losses in the filter cake due to binding onto Aerosil is increased
with high amounts of Aerosil, although this effect was somewhat offset by
a decrease in the loss of IgG in the Ppt G supernatant. Significantly,
however, amidolytic activity could not be reduced by high amounts of
Aerosil when the II+III paste is extracted at pH 4.5. Furthermore,
although γ-globulin purity is improved with higher amounts of
Aerosil, in all cases it is still below the specification limit of
>86% for Ppt G. likely because of the low pH at the extraction and
clarification.

[0223] The present example demonstrates the effect of the extraction
buffer pH on the removal of impurities following II+III paste
re-suspension and clarification.

[0224] Low pH extraction of modified II+III paste was performed at pH 4.2
using a ratio of 15 grams buffer per gram II+III paste. The suspension
was then split into 3 parts and the pH adjusted to 4.5, 4.7, or 5.0
respectively with 3M Tris. Afterwards, each solution was further split
into two parts, which were incubated for one hour at either 4° C.
or 25° C. Filtration with Cuno 50(90)SA was performed using a 10
mM sodium acetate post-wash buffer having the same pH as the respective
clarification buffer. The filtrates were treated with 8 g L-1
citrate and 0.2% Tween 80, and then IgG was precipitated by addition of
25% ethyl alcohol at -10° C. for at least 8 hours. The precipitate
was recovered by centrifugation as described previously, and the
precipitate was dissolved into a 2-fold volume of purified water. The
resulting suspension was filtered using a Cuno VR06 filtration devise, in
a final solution having a conductivity of about 1.3 mS cm-1.

[0225] To evaluate the various conditions, the level of IgG, IgA, IgM,
transferrin, fibrinogen, and other impurities were determined. The
results of this analysis are given in Table 7, which shows the pH
dependency of clarification of II+III paste suspension. Within the pH
range of 4.5 to 5.0 IgA, IgM, transferring, and fibrinogen amount does
not vary in a wide range, but other unwanted proteins are present at a
higher levels when the lower pH buffers are used. It was calculated that
these other impurities comprise 10% of the total protein at pH 4.5, but
less than 1% at pH 5. The IgG content is the highest at pH 5.0, while the
temperature dependence of IgG content and impurity levels, between
4° C. and 25° C., is negligible.

[0226] As can be seen in Table 8, further analysis of the dissolved Ppt G
fraction and VR06-filtrate indicate that use of buffers having a pH of
4.5 for the II+III clarification and II+III filtrate treatment steps,
results in increased level of aggregates and low molecular weight
components. This effect is further enhanced when the steps are performed
at 25° C., rather than 4° C. Conversely, when the samples
are treated at higher pH (pH 5.0), the resulting Ppt G suspensions and
filtrate contain higher levels of ˜350 kDa material (dimeric IgG or
IgA) than do the solutions treated at pH 4.5 (Table 8). Furthermore, the
lower pH treatments resulted in higher amidolytic activity levels than
did treatment at pH 5.0.

[0227] The results presented in Tables 7 and 8 show that the re-suspension
of II+III precipitate should be performed at refrigerated temperatures
(2° C. to 8° C.) and that pH should be kept at pH 5.0 in
order to minimize dissolution of high (>450 KDa) and low (>70 KDa)
molecular weight components, as well as components having amidolytic
activity. Effective clarification after II+III paste suspension will
reduce the impurity load for chromatography downstream processing of Ppt
G and is therefore key for meeting the IVIG final container
specifications reproducibly. To further validate this finding, modified
II+III paste was dissolved and processed, as above, the modified II+III
paste was dissolved at pH 4.2 and then adjusted to pH of 4.5, 4.7, or
5.0. As seen in Table 9, IgM is removed more efficiently by a pH of 5.0
during II+III paste suspension and clarification than removal at pH 4.5.
In this experiment IgG yield is similar at pH 5.0 and 4.5.

[0228] To evaluate the pH optimum at the II+III paste extraction step, for
minimized proteolytic activities in the filtrate, pH during extraction
and filtration was varied in a wider range from pH 3.8 to 7.8. For this
purpose modified II+III paste was extracted at low pH in a ratio of 1+8.
After a short time of stirring, to obtain a homogeneous dispersion, the
suspension was divided into 8 parts, the pH adjusted with acetic acid or
Tris buffer to either pH 3.8, 4.2, 4.6, 5.0, 6.6, 7.0, 7.4 or 7.8, and
extracted for an additional 120 minutes. Afterwards, pH was adjusted to
5.1 and clarification was done by centrifugation in 50 mL Falcon tubes.
Ppt G precipitation was performed under standard conditions. Amidolytic
activity and PKA was measured in the Ppt G dissolved fraction as
indicated in FIGS. 5 and 6.

[0229] As can be seen in the sample stored at 4° C. in FIG. 5,
amidolytic activity is minimized when the II+III paste is extracted at pH
5.0. Further emphasizing the point that the samples should not be kept at
elevated (room temperature) for extended periods of time, amidolytic
activity was elevated after storage of the Ppt G dissolved fraction at
room temperature for one week. Similarly, as seen in FIG. 6, PKA activity
is minimized when the II+III paste is extracted at pH 5.0 or higher.

Example 10

[0230] The present example evaluates the pH dependency on IgG yield loss
during extraction and clarification. Briefly, 110 grams of modified
II+III paste was re-suspended at ratio of 15 grams purified water per
gram II+III paste, followed by extraction for 120 minute. The sample was
then divided into four parts and the pH adjusted with acetic acid to pH
3.8, 4.2, 4.6, or 5.0. Pre-extraction was done before dividing into four
parts at native pH to ensure four identical parts which were then
adjusted to the mentioned pH and further extracted for one additional
hour. Samples were then clarified by Cuno 50SA filtration at the same pH
used for each extraction and Ppt G precipitation. After Cuno filtration,
all parts were treated the same way, which means standard precipitate G
precipitation at pH 7.0 for all parts. The results of two such
experiments are summarized below in Tables 10 and 11.

[0231] The data above confirms the results shown in FIGS. 5 and 6
concerning the activation of proteolytic enzymes by low pH extraction.
Taken together, the data demonstrate that low pH extraction causes less
IgG loss due to the increased solubility of all proteins. These results
are consistent with the examples provided above. Additionally, the IgG
losses shown in Table 11 suggest that higher concentrations of acetic
acid, resulting in lower pH, results in less IgG loss in the filter cake
but higher IgG loss in Precipitate G supernatant. This phenomenon might
be explained by the higher acetate concentrations at the precipitation
step after low pH extraction.

Example 11

[0232] To determine suitable II+III extraction conditions, a protocol
employing low pH extraction with purified water adjusted with acetic acid
to pH 4.3 and readjustment to pH 4.9 before filtration was compared with
extraction in 5 mM sodium acetate/5 mM sodium di-hydrogen phosphate with
600 g acetic acid per 1000 liter of extraction buffer, resulting in a
dissolution pH of 4.8 to 4.9. The experiments were performed in pilot
scale, starting with 3.8 to 5 kg of modified II+III paste. All
experiments included Aerosil treatment with 40 g Aerosil per kg II+III
paste. For clarification, a Strassburger filter press with filter frames
of 30 cm*30 cm equipped with Cuno 50SA filter sheets was used. Post wash
was performed with 4 dead volumes of the filter press, with a 5 mM sodium
acetate/5 mM sodium di-hydrogen phosphate buffer with 150 gram acetic
acid per 1000 liter of buffer. Centrifugation of precipitate G was
performed with a Cepa® Z61H centrifuge at 17000 RPM (rotor diameter
of 10.5 cm) at a flow rate of 40 liter per hour. The results of the
experiment, done in triplicate, is shown in Table 12.

[0233] As seen in Table 12, both extraction protocols result in similar
IgG recoveries. Given the results of Examples 3 through 10, which show
that various impurities can be minimized by extraction of II+III paste at
pH 5.0, the results provided in Table 12 show that extraction at pH 4.8
to 4.9 with 600 g glacial acetic acid per 1000 L of extraction buffer is
superior to extraction at low pH and subsequent adjustment to pH 4.8 to
5.0.

Example 12

[0234] During manufacturing, filter press frames and lines connecting to
and from the tanks have a significant void volume, which is still filled
with suspension or filtrate before post-wash is started. When the post
wash is finished this void volume remains in the filter press. Standard
protocols account for this void volume by washing with between one and
two dead volumes of the filter press. In order to determine if standard
post-filter washes allow for efficient recovery of total IgG, experiments
using varying volumes of wash buffer were performed on large-scale
manufacturing IgG purifications. FIG. 1 shows the dependency of post-wash
volumes (as measured by dead or void volumes) on the levels of residual
IgG and total protein.

[0235] Notably, as seen in FIG. 1, a post-filtration wash using about
2-fold filter press dead volume results in significant loss of IgG
recovery, as the wash solution contains about 1.5 gram IgG per L wash
solution. Surprisingly, it was found that 3.6-fold filter press dead
volume of post-wash buffer is required for efficient recovery of IgG
(denoted by arrow in FIG. 1). Further post-washing beyond 3.6 dead
volumes of the filter press is not expedient, as it will lead to dilution
of the filtrate without additional IgG recovery.

Example 13

[0236] During the II+III precipitation step, alcohol concentration was
increased from 20 to 25% and the temperature was lowered from -5°
C. to -7° C. When dissolving the II+III paste, at least 600 mL
glacial acetic acid was used per 1000 L volume to adjust pH of II+III
paste re-suspension buffer, in contrast to previously use ratio of 510 mL
glacial acetic acid/1000 L buffer. The extraction ratio was 1+15 with the
acetic acid buffer. For clarification, 0.04 to 0.06 gram of Aerosil
(typically at the low end of this range, e.g., 0.04 g) was added for each
gram of II+III paste. For post-wash, about four (4×) filter press
dead volumes of post-wash buffer was used. For example, 4.3× filter
press dead volumes was used in one particular experiment whereas
3.6× volumes was used in another experiment. The four times or more
dead volumes post-wash was increased from previously used 1.8× dead
volumes. The buffer was adjusted with 150 mL glacial acetic acid was used
per 1000 L buffer, an increase from previously 120 mL glacial acetic
acid/1000 L buffer. These changes led to an 8% higher yield of IgG and a
purity of at least 86% γ-globulin. Very low residual amount of IgG
was found in the filter cake extract, when extracted with 0.1 M sodium
phosphate +150 mM NaCl (pH 7.4, conductivity 25.5 mS/cm).

[0237] Table 13 shows IgG yield by the manufacturing methods currently in
use and provides a comparison reference in the experiments described
below. 15-20% of IgG is lost from Cohn pool to filtrate. About 0.4 g IgG
per liter plasma is lost in the II+III supernatant.

[0238] Cohn pool was thawed at 24-27° C. for 6-7 hours in a
14-liter bucket. Afterwards the material was mixed overnight at
2-8° C. The pool was then divided into four parts (800 g each):
[0239] 1: Fluent-wise ethanol addition, followed by pH adjustment to 7.0
[0240] 2: Fluent-wise ethanol addition, followed by pH adjustment to 7.5
[0241] 3. Ethanol addition by spraying, followed by pH adjustment to 7.0
[0242] 4. Ethanol addition by spraying, followed by pH adjustment to 7.5

[0243] All parts were first cooled to 0° C. 8% ethanol was then
added to parts 1 and 2 fluent-wise and by spraying to parts 3 and 4 using
a spray head. In both methods ethanol was added at approximately the same
speed. During ethanol addition, the cryostat was adjusted to -5°
C. and 75 ml ethanol was added to each part while mixing. The pH was
adjusted to either 7.0 or 7.5 by 1 M acetic acid. The solution was then
incubated for 1 hour. After the incubation the solution was centrifuged
with a beaker centrifuge (4600 rpm; 30 min; -2° C.).

Results

[0244] IgG yields were measured nephelometrically and are shown in Table
14. Almost 100% IgG yield in the fractionation I supernatant was obtained
with the improved method (ethanol spraying) while with conventional
ethanol addition 0.2 to 0.25 g/L plasma was lost. These results indicate
that the improved method may lead to an increase of IgG yield of up to
0.2 g/L plasma in manufacturing.

[0245] 2.8 kg plasma was thawed while mixing at 2° C. Fraction I:
8% ethanol was added and the pH was adjusted to 7.4 using 5 M acetic
acid. While mixing, the suspension was cooled to a temperature of
-2° C. Spraying conditions were obtained using a spray head. In
both methods ethanol and 5 M acetic acid addition was performed at
approximately the same speed. After 1 hour incubation, the solution was
centrifuged using a CEPA centrifuge at a temperature of -4° C.

[0246] Fraction II+III: pH was adjusted to 6.7 using a pH 4 buffer, then
25% ethanol was added (1) by spraying or (2) by fluent-wise as
conventionally performed. The pH was then readjusted to 6.9. Incubation
was conducted for 10 hours at -7° C.

Results

[0247] IgG loss during fraction II+III at 25% ethanol was measure
nephelometrically and is shown in Table 15. The IgG measurements had a
certain variation, the average value of the optimized method were
therefore taken.

[0248] Up to the point of fraction II+III precipitate only 0.35 g IgG/L
plasma was lost. A yield increase of 0.04 g IgG per liter plasma during
fractionation II+III using spraying method was achieved, compared to 25%
ethanol addition fluent-wise; and a yield increase of 0.3 g IgG per liter
plasma was achieved (averaged from the range of 0.4 to 0.06 g/L),
compared to 20% ethanol addition fluent-wise as currently used in
manufacturing. The IgG yield in the filtrate is significantly higher
compared to the reference and far above the 80 to 86% achieved currently
in manufacturing with addition of 20% ethanol fluent-wise at II+III
precipitation.

[0249] 50 liter plasma was thawed while mixing at 17-20° C. for 27
hours. Fractionation I was performed as mentioned in the above sections
as the optimized process. Supernatant I was separated into two parts:
[0250] (1) worst case pH adjustment: pH adjustment before and after
ethanol addition but not during incubation period. [0251] (2) optimized
pH adjustment: pH adjustment before and after ethanol addition and
further readjustment of the pH during hold time. The solution was
constantly stirred during hold time.

[0252] pH of the supernatant of I was adjusted in both parts to 6.7 before
ethanol addition using pH 4 buffer. Ethanol was added by spraying and pH
was readjusted to 6.9 after ethanol addition.

[0253] In part (1) the pH adjustment was carried out with less care to
simulate a worst case scenario. pH of the solution was adjusted directly
after ethanol addition but not during the incubation. In part (2) the pH
was readjusted to a constant value of 6.8 to 7.0 during incubation time
of 10 hours.

Results

[0254] IgG was again measure nephelometrically and is shown in Table 16.
By constant readjustment of the pH to a constant value of about 6.9
during the hold time, only 0.13 g IgG per liter plasma was lost compared
to an average of 0.4 g/L plasma in large-scale manufacturing. Yield
increase of 0.07 g IgG per liter plasma was achieved in comparison to
reference (without spraying but with constant stirring during hold
period). Yield increase of about 0.20 g to about 0.30 g IgG per liter
plasma was achieved compared to the conventional method currently in use
(loss of 0.38 g IgG per liter plasma, see Table 13).

[0255] IgG loss in II+III supernatant is reduced from the current level of
0.4 g IgG/L plasma in manufacturing batches to a level of 0.13 g/L plasma
at precipitation with 20% ethanol, and to a level of less than 0.08 g/L
plasma at precipitation with 25% ethanol when ethanol is added by
spraying and a continuous pH of 6.9±0.1 is maintained during
precipitation.

[0256] At precipitation I, ethanol addition before pH adjustment by
spraying leads to an IgG yield increase of 0.1 to 0.2 g/L plasma in
fractionation I supernatant.

Discussion

[0257] IgG was measured nephelometrically in all experiments and can have
a variance of at least -/+5.0% (as indicated by the manufacturer of the
nephelometer, Siemens AG). It is therefore possible that the actual yield
increase obtained by the improved method during manufacturing may be
slightly lower or higher than indicated in the examples.

[0258] As additional proof of the yield increase by the new and improved
method, the precipitate IgG weight was compared to the average
precipitate IgG weight obtained from the same plasma source in
manufacturing. 18 kg precipitate IgG is obtained per 1000 liter US source
Cohn Pool by the method currently used in manufacturing, in contrast to
the pilot scale study (section B above) where 20.8 kg precipitate IgG was
obtained (20% ethanol and optimized pH adjustment at fractionation
II+III, all buffer and ethanol addition by spraying). This is an increase
of more than 2 kg precipitate IgG per 1000 liter Cohn Pool.

Example 17

[0259] This example demonstrates that the addition of a fumed silica
treatment step prior to filtration of the Fraction II+III suspension
results in higher purity IgG filtrates. Briefly, cryo-poor plasma was
fractionated as described above to the Fraction II+III stage, at which
point it was split into two samples. The first sample was clarified only
by addition of filter aid prior to standard Fraction II+III suspension
filtration (FIG. 7A). the second sample was subjected to fumed silica
pretreatment, as described herein, prior to addition of filter aid and
standard Fraction II+III suspension filtration (FIG. 7B).

[0260] The protein components of the filtrates were then separated by
cellulose acetate electrophoresis and the areas of the individual peaks
were calculated using standard methods. As can be seen in the
chromatographs and quantitated data, the second sample, which was treated
with fumed silica prior to filtration, resulted in a filtrate with a much
higher IgG purity than the sample not treated with fumed silica (68.8%
vs. 55.7 γ-globulin; compare Table 18 with Table 16).

[0261] The present example illustrates ultrafiltration and formulation of
a 20% IgG preparation suitable for subcutaneous administration. This
information was gathered during production of scale-up and pre-clinical
20% IgG preparations. The process used for manufacturing of 20% lots
prior to the nanofiltration step was as described above.
Ultra-/diafiltration was improved to concentrate the solution to 20%. In
order to reduce yield loss to a minimum, the post-wash of the
ultrafiltration device used for diafiltration is concentrated by a second
smaller device equipped with the same membranes and afterwards added to
the bulk solution.

[0262] Surprisingly it could be shown that virus inactivation during low
pH storage is not influenced by the protein concentration of the
solution. Similar virus reduction was achieved in both 10% solution
(GAMMAGARD® LIQUID) and in 20% solution. Therefore low pH storage as
a virus reduction step was maintained for the 20% product.

[0263] Prior to nanofiltration, the glycine concentration of the IgG
solution is adjusted to a target of 0.25M. The solution is then
concentrated to a protein concentration of 6±2% w/v through
ultrafiltration (UF). The pH is adjusted to 5.2±0.2. The UF membrane
used has a Nominal Molecular Weight Cut Off (NMWCO) of 50,000 daltons or
less and is especially designed for high viscosity products (e.g., V
screen from Millipore).

[0264] The concentrate is then diafiltered against a 0.25M glycine
solution, pH 4.2±0.2. The minimum exchange volume is 10 times the
original concentrate volume. Throughout the ultrafiltration/diafiltration
operation, the solution is maintained at between about 4° C. to
20° C.

[0265] After diafiltration, the solution is concentrated to a protein
concentration of at least 22% (w/v). The solution temperature is adjusted
to 2° C. to 8° C.

[0266] In order to recover the complete residual protein in the system,
the post-wash of the first bigger ultrafiltration system is done with at
least 2 times the dead volume in re-circulation mode to assure that all
protein is washed out. Then the post-wash of the first ultrafiltration
system is concentrated to a protein concentration of at least 22% w/v
with a second ultra-/diafiltration system equipped with the same type of
membrane which is dimensioned a tenth or less of the first one. The
post-wash concentrate is added to the bulk solution. The second
ultrafiltration system is then post-washed. This post-wash is used for
adjustment of the protein concentration of the final formulation. The
solution temperature is maintained at between about 2° C. to
8° C.

[0267] In order to formulate the final solution, the protein concentration
is adjusted to about 20.4±0.4% (w/v) with post-wash of the second
smaller ultrafiltration system and/or with diafiltration buffer. The pH
is adjusted to between about 4.4 to 4.9, if necessary.

Example 19

[0268] In order to compare the fraction of IgG recovered in the Fraction
II+III filtrate in the current GAMMAGARD® LIQUID manufacturing
process, five manufacturing scale purifications of IgG were performed
using the improved Fraction II+III precipitation and dissolution methods
provided herein. Briefly, precipitation of IgG Cohn pools with a starting
IgG concentration of about 6.14 g/L was performed at -7° C. with
25% ethanol incorporated by fluent addition, as compared to -5° C.
and 20% ethanol, as employed in the current manufacturing process. The
modified Fraction II+III precipitate was then extracted 1 to 15 with a
dissolution buffer having a pH of 4.3 or adjusted with 0.06% glacial
acetic acid, and subsequently filtered through a depth filter with a
final wash of 4.3 dead filter volumes of dissolution buffer. As seen in
Table 18, modified Fraction II+III filtrate prepared according to the
improved methods provided herein contained a significantly higher
percentage (at least an 8.0% increase) of the IgG present in the starting
Cohn pool than did Fraction II+III filtrate prepared according to the
present manufacturing procedure (91.1% and 91.6% vs. 83.1% and 83.8%,
respectively).

[0269] In order to determine the purity of the IgG compositions provided
herein, three lots of IgG were prepared according to the improved methods
provided herein. The final IgG products of these purifications were then
tested for several contaminants, including IgA, IgM, Amidolytic activity,
C3, and fibrinogen, as well as to determine the percentage of IgG
monomers/dimers in the final composition. As can be seen in Table 19
below, the improve methods manufacture provided herein result in final
bulk compositions with increased IgG recovery, 73.6% to 78.5% of the
starting material as compared to 60% to 70% for currently employed
methods, while maintaining purity profiles that are as good, if not
better, than current IgG manufacting standards.

[0270] It is understood that the examples and embodiments described herein
are for illustrative purposes only and that various modifications or
changes in light thereof will be suggested to persons skilled in the art
and are to be included within the spirit and purview of this application
and scope of the appended claims. All publications, patents, and patent
applications cited herein are hereby incorporated by reference in their
entirety for all purposes.